Electrokinetically-altered fluids comprising charge-stabilized gas-containing nanostructures

ABSTRACT

Provided are electrokinetically-altered fluids (e.g., gas-enriched (e.g., oxygen-enriched) electrokinetic fluids) comprising an ionic aqueous solution of charge-stabilized gas-containing nanostructures in an amount sufficient to provide, upon contact with a cell, modulation of at least one of cellular membrane potential and cellular membrane conductivity. Further provided are the methods of making the electrokinetically-altered ionic aqueous fluid compositions. Particular aspects provide for regulating or modulating intracellular signal transduction associated by modulation of at least one of cellular membranes, membrane potential, membrane proteins such as membrane receptors, including but not limited to G-Protein Coupled Receptors (GPCR), and intercellular junctions (e.g., tight junctions, gap junctions, zona adherins and desmasomes). Other embodiments include particular methods of producing the electrokinetically-altered fluids. The electrokinetically-altered fluid compositions and methods of producing the fluid include electrokinetically-altered ionic aqueous fluids optionally in the form of solvated electrons stabilized, at least in part, with molecular gas (e.g., oxygen).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed generally to mixing devices and moreparticularly to mixing devices that mix two or more materials betweensurfaces, including such as between a rotating rotor and a stationarystator. Particular aspects relate to electrokinetically-altered fluids(e.g., gas-enriched (e.g., oxygen-enriched) electrokinetic fluids)comprising an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures in an amount sufficient to provide,upon contact with a cell, modulation of at least one of cellularmembrane potential and cellular membrane conductivity. Further aspectsprovide for the method of making the electrokinetically-altered ionicaqueous fluid compositions. Particular aspects provide for regulating ormodulating of at least one of cellular membrane potential, cellularmembrane conductivity, and intracellular signal transduction associatedby modulation of at least one of cellular membranes, membrane potential,membrane proteins such as membrane receptors, including but not limitedto G-Protein Coupled Receptors (GPCR), and intercellular junctions(e.g., tight junctions, gap junctions, zona adherins and desmasomes).Other embodiments include particular methods of producing theelectrokinetically-altered fluids, wherein the biological effects of theinventive fluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the ionic components of the fluids, and/or by alteringthe gas component of the fluids.

2. Description of the Related Art

FIG. 1 provides a partial block diagram, partial cross-sectional view ofa prior art device 10 for diffusing or emulsifying one or two gaseous orliquid materials (“infusion materials”) into another gaseous or liquidmaterial (“host material”) reproduced from U.S. Pat. No. 6,386,751,incorporated herein by reference in its entirety. The device 10 includesa housing configured to house a stator 30 and a rotor 12. The stator 30encompasses the rotor 12. A tubular channel 32 is defined between therotor 12 and the stator 30. The generally cylindrically shaped rotor 12has a diameter of about 7.500 inches and a length of about 6.000 inchesproviding a length to diameter ratio of about 0.8.

The rotor 12 includes a hollow cylinder, generally closed at both ends.A gap exists between each of the first and second ends of the rotor 12and a portion of the housing 34. A rotating shaft 14 driven by a motor18 is coupled to the second end of the rotor 12. The first end of therotor 12 is coupled to an inlet 16. A first infusion material passesthrough the inlet 16 and into the interior of the rotor 12. The firstinfusion material passes from the interior of the rotor 12 and into thechannel 32 through a plurality of openings 22 formed in the rotor 12.

The stator 30 also has openings 22 formed about its circumference. Aninlet 36 passes a second infusion material to an area 35 between thestator 30 and the housing 34. The second infusion material passes out ofthe area 35 and into the channel 32 through openings 22.

An external pump (not shown) is used to pump the host material into asingle inlet port 37. The host material passes through a single inletport 37 and into the channel 32 where it encounters the first and secondinfusion materials, which enter the channel 32 through openings 22. Theinfusion materials may be pressurized at their source to prevent thehost material from passing through openings 22.

The inlet port 37, is configured and positioned such that it is locatedalong only a relatively small portion (<about 5%) of the annular inletchannel 32, and is substantially parallel to the axis of rotation of therotor 12 to impart an axial flow toward a portion of the channel 32 intothe host material.

Unfortunately, before entering the tubular channel 32, the host materialmust travel in tortuous directions other than that of the axial flow(e.g., including in directions substantially orthogonal thereto) anddown into and between the gap formed between the first end of the rotor12 and the housing 34 (i.e., down a portion of the first end of therotor adjacent to the inlet 16 between the end of the rotor 12 and thehousing 34). The non-axial and orthogonal flow, and the presence of thehost material in the gap between the first end of the rotor 12 and thehousing 34 causes undesirable and unnecessary friction. Further, it ispossible for a portion of the host material to become trapped in eddycurrents swirling between the first end of the rotor and the housing.Additionally, in the device 10, the host material must negotiate atleast two right angles to enter any aspect of the annual of the annularinlet of the tubular channel 32.

A single outlet port 40 is formed in the housing 34. The combined hostmaterial and infusion material(s) exit the channel 32 via the outlet 40.The outlet port 40, which is also located along only a limited portion(<about 5%) of the annular outlet of tubular channel 32, issubstantially parallel to the axis of rotation of the rotor 12 to impartor allow for an axial flow of the combined materials away from thelimited portion of the annular outlet of tubular channel 32 into theoutlet port 40. An external pump 42 is used to pump the exiting fluidthrough the outlet port 40.

Unfortunately, before exiting the channel 32, a substantial portion ofthe exiting material must travel in a tortuous direction other than thatof the axial flow (e.g., including in directions substantiallyorthogonal thereto) and down into and between the gap formed between thesecond end of the rotor 12 and the housing 34 (i.e., down a portion ofthe second end of the rotor adjacent to the shaft 14 between the end ofthe rotor 12 and the housing 34). As mentioned above, the non-axial andorthogonal flow, and the presence of the host material in the other gapbetween the end (in this case, the second end) of the rotor 12 and thehousing 34 causes additional undesirable and unnecessary friction.Further, it is possible for a portion of the host material to becometrapped in eddy currents swirling between the second end of the rotorand the housing. Additionally, in the device 10, a substantial portionof the exiting combined material must negotiate at least two rightangles as it exits form the annular exit of the tubular channel 32 intothe outlet port 40.

As is apparent to those of ordinary skill in the art, the inlet port 37imparts only an axial flow to the host material. Only the rotor 21imparts a circumferential flow into the host material. Further, theoutlet port 40 imparts or provides for only an axial flow into theexiting material. Additionally, the circumferential flow velocity vectoris imparted to the material only after it enters the annular inlet 37 ofthe tubular channel 32, and subsequently the circumferential flow vectormust be degraded or eliminated as the material enters the exit port 40.There is, therefore, a need for a progressive circumferentialacceleration of the material as it passes in the axial direction throughthe channel 32, and a circumferential deceleration upon exit of thematerial from the channel 32. These aspects, in combination with thetortuous path that the material takes from the inlet port 37 to theoutlet port 40, create a substantial friction and flow resistance overthe path that is accompanied by a substantial pressure differential (26psi, at 60 gallons/min flow rate) between the inlet 37 and outlet 40ports, and these factors, inter alia, combine to reduce the overallefficiency of the system.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is a partial cross-section, partial block diagram of a prior artmixing device.

FIG. 2 is block diagram of an exemplary embodiment of a mixing device.

FIG. 3 is an illustration of an exemplary system for delivering a firstmaterial to the mixing device of FIG. 2.

FIG. 4 is a fragmentary partial cross-sectional view of a top portion ofthe mixing device of FIG. 2.

FIG. 5 is a fragmentary cross-sectional view of a first side portion ofthe mixing device of FIG. 2.

FIG. 6 is a fragmentary cross-sectional view of a second side portion ofthe mixing device of FIG. 2.

FIG. 7 is a fragmentary cross-sectional view of a side portion of themixing device of FIG. 2 located between the first side portion of FIG. 5and the second side portion of FIG. 6.

FIG. 8 is a perspective view of a rotor and a stator of the mixingdevice of FIG. 2.

FIG. 9 is a perspective view of an inside of a first chamber of themixing device of FIG. 2.

FIG. 10 is a fragmentary cross-sectional view of the inside of a firstchamber of the mixing device of FIG. 2 including an alternate embodimentof the pump 410.

FIG. 11 is a perspective view of an inside of a second chamber of themixing device of FIG. 2.

FIG. 12 is a fragmentary cross-sectional view of a side portion of analternate embodiment of the mixing device.

FIG. 13 is a perspective view of an alternate embodiment of a centralsection of the housing for use with an alternate embodiment of themixing device.

FIG. 14 is a fragmentary cross-sectional view of an alternate embodimentof a bearing housing for use with an alternate embodiment of the mixingdevice.

FIG. 15 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor approaches (but is not aligned with) anaperture of the stator.

FIG. 16 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen the through-hole of the rotor is aligned with the aperture of thestator.

FIG. 17 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor that was previously aligned with theaperture of the stator is no longer aligned therewith.

FIG. 18 is a side view of an alternate embodiment of a rotor.

FIG. 19 is an enlarged fragmentary cross-sectional view taken through aplane orthogonal to an axis of rotation of the rotor depicting analternate configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 20 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting a configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 21 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting an alternate offset configuration of through-holesformed in the rotor and through-holes formed in the stator.

FIG. 22 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 23 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 24 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 25 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 26 is an illustration of an electrical double layer (“EDL”) formednear a surface.

FIG. 27 is a perspective view of a model of the inside of the mixingchamber.

FIG. 28 is a cross-sectional view of the model of FIG. 27.

FIG. 29 is an illustration of an experimental setup.

FIG. 30 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored a 500 ml thin walledplastic bottle and a 1,000 ml glass bottle each capped at 65°Fahrenheit.

FIG. 31 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored in a 500 ml plasticthin walled bottle and a 1,000 ml glass bottle both refrigerated at 39°Fahrenheit.

FIG. 32 illustrates the dissolved oxygen retention of a 500 ml beveragefluid processed with oxygen in the mixing device of FIG. 2.

FIG. 33 illustrates the dissolved oxygen retention of a 500 ml braunbalanced salt solution processed with oxygen in the mixing device ofFIG. 2.

FIG. 34 illustrates a further experiment wherein the mixing device ofFIG. 2 is used to sparge oxygen from water by processing the water withnitrogen in the mixing device of FIG. 2.

FIG. 35 illustrates the sparging of oxygen from water by the mixingdevice of FIG. 2 at standard temperature and pressure.

FIG. 36 is an illustration of an exemplary nanocage.

FIGS. 37A and B illustrate Rayleigh scattering effects of anoxygen-enriched fluid;

FIG. 38 illustrates the cytokine profile of a mitogenic assay in thepresence of a gas-enriched fluid and deionized control fluid; and

FIG. 39 illustrates the difference in the growth rates of Pseudomonasbacteria at various dissolved oxygen saturation ratios.

FIGS. 40A and 40B illustrate in vitro healing of wounds using anoxygen-enriched cell culture media and a non-gas-enriched media.

FIGS. 41A through 41F show histological cross-sections of dermal andepidermal in vivo wound healing.

FIG. 42 illustrates the expression of Hale's stain in treated andcontrol healing wounds, used to detect acid mucopolysaccharides, such ashyaluronic acid;

FIG. 43 illustrates the expression of von Willebrand's Factor stain usedto detect angiogenesis in treated and control healing wounds;

FIG. 44 illustrates the detection of Luna's stain used to detect elastinin treated and control healing wounds;

FIG. 45 illustrates the number of mast cells per visual field fortreated and control healing wounds;

FIG. 46 illustrates the percentage of dead cells at separate time pointsin a corneal fibroblast assay using inventive gas-enriched culture mediaand control culture media,

FIG. 47 illustrates the shelf life of the inventive gas-enriched fluidin a polymer pouch;

FIG. 48 illustrates the results of contacting splenocytes with MOG inthe presence of pressurized pot oxygenated fluid (1), inventivegas-enriched fluid (2), or control deionized fluid (3).

FIGS. 49-58 show the results of whole blood sample evaluations ofcytokines.

FIGS. 59-68 show the corresponding cytokine results of bronchoalveolarlavage fluid (BAL) sample evaluations.

FIGS. 69-75 shows studies where the Bradykinin B2 membrane receptor wasimmobilized onto aminopropylsilane (APS) biosensor. The Sample plate setup was as designated in FIG. 69 and the binding of Bradykinin to theimmobilized receptor was assessed according to the sample set up asdesignated in FIG. 71. Results of Bradykinin binding are shown in FIG.72. Bradykinin binding to the receptor was further titrated according tothe set-up as designated in FIG. 73. As indicated in FIG. 74, Bradykininbinding to the B2 receptor was concentration dependent, and bindingaffinity was increased in the proprietary gas-enriched saline fluid ofthe instant disclosure compared to normal saline. Stabilization ofBradykinin binding to the B2 receptor is shown in FIG. 75.

FIGS. 76-83 show data showing the ability of particular embodimentsdisclosed herein to affect regulatory T cells. The study involvedirradiating antigen presenting cells, and introducing antigen and Tcells.

FIG. 84 shows that the inventive electrokinetically generated fluidsdecreased serum uptake of salmon calcitonin and an animal model. Theresults are consistent with enhancement of tight junctions.

FIGS. 85-89 show the expression levels of tight junction-relatedproteins in lung tissue from the animal model used to generate the dataof FIG. 84.

FIGS. 90-94 show data obtained from human foreskin keratinocytes exposedto RDC1676-01 (sterile saline processed through the instant proprietarydevice with additional oxygen added; gas-enriched electrokineticallygenerated fluid (Rev) of the instant disclosure) showing up-regulationof NOS1 and 3, and Nostrin, NOS3.

FIGS. 95 and 96 show data supporting localized electrokinetic effects(voltage/current) occurring in a mixing device comprising insulatedrotor and stator features to allow for detection of voltage/currenteffects during electrokinetic fluid generation.

FIGS. 97A-C show results of nuclear magnetic resonance (NMR) studiesconducted to further characterize the fundamental nature of theinventive electrokinetically generated fluids. The electrokineticallygenerated fluids increased the ¹³C-NMR line-widths of the reporterTrehalose solute.

FIGS. 98 and 99 show results of voltametric studies (i.e., square wavevoltametry (FIG. 98) and stripping polarography (FIG. 99)) conducted tofurther characterize the fundamental nature of the inventiveelectrokinetically generated fluids. Square wave voltametry peakdifferences (with respect to control) unique to the electrokineticallygenerated fluids were observed at −0.14V, −0.47V, −1.02V and −1.36V.Pronounced polaragraphic peaks were seen at −0.9 volts for theelectrokinetically generated Revera and Solas fluids, and the spectra ofthe non-electrokinetically generated blank and saline control fluidsshow characteristic peaks at −0.19 and −0.3 volts that are absent in thespectra for the electrokinetically generated fluids.

FIGS. 100-106 show results of patch clamping techniques that assessedthe effects of the electrokinetically generated fluid test on epithelialcell membrane polarity and ion channel activity. The results indicatethat the inventive electrokinetically generated fluids affect avoltage-dependent contribution of the whole-cell conductance.

FIGS. 107A-D and 108A-D show data indicating that the inventiveelectrokinetically generated fluids (e.g., RDC1676-00, RDC1676-01,RDC1676-02 and RDC1676-03) protected against methacholine-inducedbronchoconstriction when administered alone or as diluents for albuterolsulfate in male guinea pigs.

FIGS. 109-114 show results of budesonide experiments performed to assessthe airway anti-inflammatory properties of the inventiveelectrokinetically generated fluids in a Brown Norway rat ovalbuminsensitization model. The inventive electrokinetically generated fluidsdecreased eosinophil count, showed strong synergy with Budesonide indecreasing eosinophil count, decreased Penh values, increased TidalVolume, decreased blood levels of Eotaxin, significantly enhanced theBlood levels of two major key anti-inflammatory cytokines, IL10 andInterferron gamma at 6 hours after challenge as a result of treatmentwith the inventive electrokinetically generated fluid (e.g., Rev 60)alone or in combination with Budesonide, and decreased systemic levelsof Rantes. The data show that there is a substantial synergistic effectof Budesonide 750 ug/kg and the inventive electrokinetically generatedfluids (e.g., Rev 60).

FIG. 115 shows that the inventive electrokinetically generated fluid(e.g., Revera 60 and Solas) reduced DEP-induced TSLP receptor expressionin bronchial epithelial cells (BEC) by approximately 90% and 50%,respectively, whereas normal saline (NS) had only a marginal effect.

FIG. 116 shows the inventive electrokinetically generated fluid (e.g.,Revera 60 and Solas) inhibited the DEP-induced cell surface bound MMP9levels in bronchial epithelial cells by approximately 80%, and 70%,respectively, whereas normal saline (NS) had only a marginal effect.

FIGS. 117 A-C demonstrate the results of a series of patch clampingexperiments that assessed the effects of the electrokineticallygenerated fluid (e.g., RNS-60 and Solas) on epithelial cell membranepolarity and ion channel activity at two time-points (15 min (leftpanels) and 2 hours (right panels)) and at different voltage protocols.

FIGS. 118 A-C show, in relation to the experiments relating to FIGS. 117A-C, the graphs resulting from the subtraction of the Solas current datafrom the RNS-60 current data at three voltage protocols (A. steppingfrom zero mV; B. stepping from −60 mV; C. stepping from −120 mV) and thetwo time-points (15 mins (open circles) and 2 hours (closed circles)).

FIGS. 119 A-D demonstrate the results of a series of patch clampingexperiments that assessed the effects of the electrokineticallygenerated fluid (e.g., Solas (panels A. and B.) and RNS-60 (panels C.and D.)) on epithelial cell membrane polarity and ion channel activityusing different external salt solutions and at different voltageprotocols (panels A. and C. show stepping from zero mV; panels B. and D.show stepping from −120 mV).

FIGS. 120 A-D show, in relation to the experiments relating to FIGS. 119A-D, the graphs resulting from the subtraction of the CsCl current data(shown in FIG. 119) from the 20 mM CaCl₂ (diamonds) and 40 mM CaCl₂(filled squares) current data at two voltage protocols (panels A. and C.stepping from zero mV; B. and D. stepping from −120 mV) for Solas(panels A. and B.) and Revera 60 (panels C. and D.).

FIG. 121A shows 1 mm2 AFM scan for RNS60-1 (rns60-1 1 um 3D.jpg). Thesmall peaks (“1”) represent hydrophobic nanobubbles which are ˜20 nmwide and ˜1.5 nm tall or smaller.

FIG. 121B shows 1 mm2 scan for PNS60-1 (pp60-1 1 um 3d.jpg). This scanreveals peaks (“2”) (hydrophobic nanobubbles) that are substantiallylarger (˜60 nm wide and ˜5 nm tall) than those visible with RNS60-1.

FIG. 122 illustrates a graphical representation of an exemplaryembodiment of a bioreactor system 3300 a.

FIG. 123 is another illustration of a graphical representation of aexemplary embodiment of a bioreactor system 3300 a.

FIG. 124 shows detailed portions of exemplary embodiments of thebioreactor system 3300 a of FIGS. 122 and 123.

SUMMARY OF EXEMPLARY EMBODIMENTS

Particular aspects provide for a electrokinetically-altered fluidcomposition, comprising an electrokinetically altered aqueous fluidcomprising an ionic aqueous solution of charge-stabilized gas-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide, upon contact of a living cell by thefluid, modulation of at least one of cellular membrane potential andcellular membrane conductivity. In certain aspects, thecharge-stabilized oxygen-containing nanostructures are the majorcharge-stabilized gas-containing nanostructure species in the fluid. Infurther aspects, the percentage of dissolved oxygen molecules present inthe fluid as the charge-stabilized oxygen-containing nanostructures is apercentage selected from the group consisting of greater than: 0.01%,0.1%, 1%, 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%;65%; 70%; 75%; 80%; 85%; 90%; and 95%. In yet further aspects, the totaldissolved oxygen is substantially present in the charge-stabilizedoxygen-containing nanostructures. According to additional aspects, thecharge-stabilized gas-containing nanostructures substantially have anaverage diameter of less than a size selected from the group consistingof: 90 nm; 80 nm; 70 nm; 60 nm; 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; andless than 5 nm. According to certain aspects, the ionic aqueous solutioncomprises a saline solution. In further aspects, the fluid issupergasified (e.g., super oxygenated, super nitrogenated, supercarbonated, etc.).

According to particular aspects, the fluid comprises at least one of aform of solvated electrons, and an electrokinetically modified orcharged gas (e.g., oxygen) species. In certain aspects, the form ofsolvated electrons or electrokinetically modified or charged gas (e.g.,oxygen) species are present in an amount of at least 0.01 ppm, at least0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5ppm, at least 7 ppm, at least 10 ppm, at least 15 ppm, or at least 20ppm. In further aspects, the electrokinetically-altered fluid comprisesa form of solvated electrons stabilized, at least in part, by the gaspresent in the nanostructure (e.g., molecular oxygen_. In furtheraspects, the ability to modulate of at least one of cellular membranepotential and cellular membrane conductivity persists for at least two,at least three, at least four, at least five, at least 6, at least 12months, or longer periods, in a closed gas-tight container.

In particular aspects, alteration of the electrokinetically alteredaqueous fluid comprises exposure of the fluid tohydrodynamically-induced, localized electrokinetic effects. According tocertain aspects, exposure to the localized electrokinetic effectscomprises exposure to at least one of voltage pulses and current pulses.According to further aspects, the exposure of the fluid tohydrodynamically-induced, localized electrokinetic effects, comprisesexposure of the fluid to electrokinetic effect-inducing structuralfeatures of a device used to generate the fluid.

According to certain aspects, modulation of at least one of cellularmembrane potential and cellular membrane conductivity comprises alteringof a conformation, ligand binding activity, or a catalytic activity of amembrane associated protein. According to further aspects, the membraneassociated protein comprises at least one selected from the groupconsisting of receptors, transmembrane receptors, ion channel proteins,intracellular attachment proteins, cellular adhesion proteins,integrins, etc. According to yet further aspects, the transmembranereceptor comprises a G-Protein Coupled Receptor (GPCR). In certainaspects, the G-Protein Coupled Receptor (GPCR) interacts with a Gprotein α subunit. In particular aspects, the G protein α subunitcomprises at least one selected from the group consisting of Gα_(s),Gα_(i), Gα_(q), and Gα₁₂. In additional aspects, at least one G proteinα subunit is Gα_(q). In further aspects, modulation of at least one ofcellular membrane potential and cellular membrane conductivity,comprises modulating whole-cell conductance. In yet further aspects,modulating whole-cell conductance, comprises modulating at least one ofa linear or non-linear voltage-dependent contribution of the whole-cellconductance.

Particular aspects provide for modulation of at least one of cellularmembrane potential and cellular membrane conductivity comprisesmodulation of a calcium dependant cellular messaging pathway or system.Additional aspects provide for modulation of at least one of cellularmembrane potential and cellular membrane conductivity comprisesmodulation of phospholipase C activity. In certain aspects, modulationof at least one of cellular membrane potential and cellular membraneconductivity comprises modulation of adenylate cyclase (AC) activity.

Particular aspects provide for electrokinetically-altered aqueous fluidcomprising dissolved gas (e.g., oxygen, or other suitable gasesincluding but not limited to gases described herein) in an amount of atleast 8 ppm, at least 15, ppm, at least 25 ppm, at least 30 ppm, atleast 40 ppm, at least 50 ppm, or at least 60 ppm oxygen at atmosphericpressure. Additional aspects provide for the gas dissolved in the fluidor solution as the nanostructures being present in an amount of at least25 ppm.

Particular aspects provide for a method of producing anelectrokinetically-altered aqueous fluid or solution, comprising:providing a flow of a fluid material between two spaced surfaces inrelative motion and defining a mixing volume therebetween, wherein thedwell time of a single pass of the flowing fluid material within andthrough the mixing volume is greater than 0.06 seconds or greater than0.1 seconds; and introducing gas (e.g., oxygen (O₂)) into the flowingfluid material within the mixing volume under conditions suitable todissolve at least 20 ppm, at least 25 ppm, at least 30, at least 40, atleast 50, or at least 60 ppm gas (e.g., oxygen) into the material, andelectrokinetically alter the fluid or solution, wherein anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized gas (e.g., oxygen)-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide, upon contact of a living cell by thefluid, modulation of at least one of cellular membrane potential andcellular membrane conductivity is provided. In additional aspects, thegas (e.g., oxygen) is infused into the material in less than 100milliseconds, less than 200 milliseconds, less than 300 milliseconds, orless than 400 milliseconds.

Certain aspects provide for an electrokinetically altered gasified(e.g., oxygenated) aqueous fluid or solution made as described herein.

Additional aspects provide for a method of producing anelectrokinetically altered aqueous fluid or solution, comprising:providing a flow of a fluid material between two spaced surfacesdefining a mixing volume therebetween; and introducing gas (e.g.,oxygen) into the flowing material within the mixing volume underconditions suitable to infuse at least 20 ppm, at least 25 ppm, at least30, at least 40, at least 50, or at least 60 ppm gas (e.g., oxygen) intothe material in less than 100 milliseconds, less than 200 milliseconds,less than 300 milliseconds, or less than 400 milliseconds, toelectrokinetically alter the fluid or solution, wherein anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized gas (e.g., oxygen)-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide, upon contact of a living cell by thefluid, modulation of at least one of cellular membrane potential andcellular membrane conductivity is provided. In certain aspects, thedwell time of the flowing material within the mixing volume is greaterthan 0.06 seconds or greater than 0.1 seconds. In further aspects, theratio of surface area to the volume is at least 12, at least 20, atleast 30, at least 40, or at least 50.

Particular aspects provide for an electrokinetically altered gasified(e.g., oxygenated) aqueous fluid or solution made according to theinvention disclosed herein.

Certain aspects provide for a method of producing anelectrokinetically-altered aqueous fluid or solution, comprising use ofa mixing device for creating an output mixture by mixing a firstmaterial and a second material, the device comprising: a first chamberconfigured to receive the first material from a source of the firstmaterial; a stator; a rotor having an axis of rotation, the rotor beingdisposed inside the stator and configured to rotate about the axis ofrotation therein, at least one of the rotor and stator having aplurality of through-holes; a mixing chamber defined between the rotorand the stator, the mixing chamber being in fluid communication with thefirst chamber and configured to receive the first material therefrom,and the second material being provided to the mixing chamber via theplurality of through-holes formed in the one of the rotor and stator; asecond chamber in fluid communication with the mixing chamber andconfigured to receive the output material therefrom; and a firstinternal pump housed inside the first chamber, the first internal pumpbeing configured to pump the first material from the first chamber intothe mixing chamber, to electrokinetically alter the fluid or solution,wherein an electrokinetically altered aqueous fluid comprising an ionicaqueous solution of charge-stabilized gas (e.g., oxygen)-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide, upon contact of a living cell by thefluid, modulation of at least one of cellular membrane potential andcellular membrane conductivity is provided.

Additional aspects provide for a method of producing anelectrokinetically altered gasified (e.g., oxygenated) aqueous fluid orsolution, comprising use of a mixing device for creating an outputmixture by mixing a first material and a second material, the devicecomprising: a stator; a rotor having an axis of rotation, the rotorbeing disposed inside the stator and configured to rotate about the axisof rotation therein; a mixing chamber defined between the rotor and thestator, the mixing chamber having an open first end through which thefirst material enters the mixing chamber and an open second end throughwhich the output material exits the mixing chamber, the second materialentering the mixing chamber through at least one of the rotor and thestator; a first chamber in communication with at least a majorityportion of the open first end of the mixing chamber; and a secondchamber in communication with the open second end of the mixing chamber,to electrokinetically alter the fluid or solution, wherein anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized gas (e.g.,oxygen)-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide, upon contact of a living cell by thefluid, modulation of at least one of cellular membrane potential andcellular membrane conductivity is provided. In certain aspects, thefirst internal pump is configured to impart a circumferential velocityinto the first material before it enters the mixing chamber.

DETAILED DESCRIPTION OF THE INVENTION Electrokinetically-GeneratedFluids

“Electrokinetically generated fluid,” as used herein, refers toApplicants' inventive electrokinetically-generated fluids generated, forpurposes of the working Examples herein, by the exemplary Mixing Devicedescribed in detail herein (see also US200802190088 and WO2008/052143,both incorporated herein by reference in their entirety). Theelectrokinetic fluids, as demonstrated by the data disclosed andpresented herein, represent novel and fundamentally distinct fluidsrelative to prior art non-electrokinetic fluids, including relative toprior art oxygenated non-electrokinetic fluids (e.g., pressure potoxygenated fluids and the like). As disclosed in various aspects herein,the electrokinetically-generated fluids have unique and novel physicaland biological properties including, but not limited to the following:

In particular aspects, the electrokinetically altered aqueous fluidcomprise an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter of less than about 100 nanometers and stably configured in theionic aqueous fluid in an amount sufficient to provide, upon contact ofa living cell by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity.

In particular aspects, electrokinetically-generated fluids refers tofluids generated in the presence of hydrodynamically-induced, localized(e.g., non-uniform with respect to the overall fluid volume)electrokinetic effects (e.g., voltage/current pulses), such as devicefeature-localized effects as described herein. In particular aspectssaid hydrodynamically-induced, localized electrokinetic effects are incombination with surface-related double layer and/or streaming currenteffects as disclosed and discussed herein.

In particular aspects the administered inventiveelectrokinetically-altered fluids comprise charge-stabilizedoxygen-containing nanostructures in an amount sufficient to providemodulation of at least one of cellular membrane potential and cellularmembrane conductivity. In certain embodiments, theelectrokinetically-altered fluids are superoxygenated (e.g., RNS-20,RNS-40 and RNS-60, comprising 20 ppm, 40 ppm and 60 ppm dissolvedoxygen, respectively, in standard saline). In particular embodiments,the electrokinetically-altered fluids are not-superoxygenated (e.g.,RNS-10 or Solas, comprising 10 ppm (e.g., approx. ambient levels ofdissolved oxygen in standard saline). In certain aspects, the salinity,sterility, pH, etc., of the inventive electrokinetically-altered fluidsis established at the time of electrokinetic production of the fluid,and the sterile fluids are administered by an appropriate route.Alternatively, at least one of the salinity, sterility, pH, etc., of thefluids is appropriately adjusted (e.g., using sterile saline orappropriate diluents) to be physiologically compatible with the route ofadministration prior to administration of the fluid. Preferably, anddiluents and/or saline solutions and/or buffer compositions used toadjust at least one of the salinity, sterility, pH, etc., of the fluidsare also electrokinetic fluids, or are otherwise compatible.

In particular aspects, the inventive electrokinetically-altered fluidscomprise saline (e.g., one or more dissolved salt(s); e.g., alkali metalbased salts (Li+, Na+, K+, Rb+, Cs+, etc.), alkaline earth based salts(e.g., Mg++, Ca++), etc., or transition metal-based positive ions (e.g.,Cr, Fe, Co, Ni, Cu, Zn, etc.,), in each case along with any suitableanion components, including, but not limited to F−, Cl−, Br−, I−, PO₄−,SO₄−, and nitrogen-based anions. Particular aspects comprise mixed saltbased electrokinetic fluids (e.g., Na+, K+, Ca++, Mg++, transition metalion(s), etc.) in various combinations and concentrations, and optionallywith mixtures of couterions. In particular aspects, the inventiveelectrokinetically-altered fluids comprise standard saline (e.g.,approx. 0.9% NaCl, or about 0.15 M NaCl). In particular aspects, theinventive electrokinetically-altered fluids comprise saline at aconcentration of at least 0.0002 M, at least 0.0003 M, at least 0.001 M,at least 0.005 M, at least 0.01 M, at least 0.015 M, at least 0.1 M, atleast 0.15 M, or at least 0.2 M. In particular aspects, the conductivityof the inventive electrokinetically-altered fluids is at least 10 μS/cm,at least 40 μS/cm, at least 80 μS/cm, at least 100 μS/cm, at least 150μS/cm, at least 200 μS/cm, at least 300 μS/cm, or at least 500 μS/cm, atleast 1 mS/cm, at least 5, mS/cm, 10 mS/cm, at least 40 mS/cm, at least80 mS/cm, at least 100 mS/cm, at least 150 mS/cm, at least 200 mS/cm, atleast 300 mS/cm, or at least 500 mS/cm. In particular aspects, any saltmay be used in preparing the inventive electrokinetically-alteredfluids, provided that they allow for formation of biologically activesalt-stabilized nanostructures (e.g., salt-stabilized oxygen-containingnanostructures) as disclosed herein.

According to particular aspects, the biological effects of the inventivefluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the ionic components of the fluids, and/or by alteringthe gas component of the fluid. According to particular aspects, thebiological effects of the inventive fluid compositions comprisingcharge-stabilized gas-containing nanostructures can be modulated (e.g.,increased, decreased, tuned, etc.) by altering the gas component of thefluid. In preferred aspects, oxygen is used in preparing the inventiveelectrokinetic fluids. In additional aspects mixtures of oxygen alongwith at least one other gas selected from Nitrogen, Oxygen, Argon,Carbon dioxide, Neon, Helium, krypton, hydrogen and Xenon. As describedabove, the ions may also be varied, including along with varying the gasconstitutent(s).

Given the teachings and assay systems disclosed herein (e.g., cell-basedcytokine assays, patch-clamp assays, etc.) one of skill in the art willreadily be able to select appropriate salts and concentrations thereofto achieve the biological activities disclosed herein.

TABLE A-1 Exemplary cations and anions. Name Formula Other name(s)Common Cations: Aluminum Al⁺³ Ammonium NH₄ ⁺ Barium Ba⁺² Calcium Ca⁺²Chromium(II) Cr⁺² Chromous Chromium(III) Cr⁺³ Chromic Copper(I) Cu⁺Cuprous Copper(II) Cu⁺² Cupric Iron(II) Fe⁺² Ferrous Iron(III) Fe⁺³Ferric Hydrogen H⁺ Hydronium H₃O⁺ Lead(II) Pb⁺² Lithium Li⁺ MagnesiumMg⁺² Manganese(II) Mn⁺² Manganous Manganese(III) Mn⁺³ ManganicMercury(I) Hg₂ ⁺² Mercurous Mercury(II) Hg⁺² Mercuric Nitronium NO₂ ⁺Potassium K⁺ Silver Ag⁺ Sodium Na⁺ Strontium Sr⁺² Tin(II) Sn⁺² StannousTin(IV) Sn⁺⁴ Stannic Zinc Zn⁺² Common Anions: Simple ions: Hydride H⁻Oxide O²⁻ Fluoride F⁻ Sulfide S²⁻ Chloride Cl⁻ Nitride N³⁻ Bromide Br⁻Iodide I⁻ Oxoanions: Arsenate AsO₄ ³⁻ Phosphate PO₄ ³⁻ Arsenite AsO₃ ³⁻Hydrogen phosphate HPO₄ ²⁻ Dihydrogen phosphate H₂PO₄ ⁻ Sulfate SO₄ ²⁻Nitrate NO₃ ⁻ Hydrogen sulfate HSO₄ ⁻ Nitrite NO₂ ⁻ Thiosulfate S₂O₃ ²⁻Sulfite SO₃ ²⁻ Perchlorate ClO₄ ⁻ Iodate IO₃ ⁻ Chlorate ClO₃ ⁻ BromateBrO₃ ⁻ Chlorite ClO₂ ⁻ Hypochlorite OCl⁻ Hypobromite OBr⁻ Carbonate CO₃²⁻ Chromate CrO₄ ²⁻ Hydrogen carbonate HCO₃ ⁻ Dichromate Cr₂O₇ ²⁻ orBicarbonate Anions from Organic Acids: Acetate CH₃COO⁻ formate HCOO⁻Others: Cyanide CN⁻ Amide NH₂ ⁻ Cyanate OCN⁻ Peroxide O₂ ²⁻ ThiocyanateSCN⁻ Oxalate C₂O₄ ²⁻ Hydroxide OH⁻ Permanganate MnO₄ ⁻

TABLE A-B Exemplary cations and anions. Formula Charge Name MonoatomicCations H⁺ 1+ hydrogen ion Li⁺ 1+ lithium ion Na⁺ 1+ sodium ion K⁺ 1+potassium ion Cs⁺ 1+ cesium ion Ag⁺ 1+ silver ion Mg²⁺ 2+ magnesium ionCa²⁺ 2+ calcium ion Sr²⁺ 2+ strontium ion Ba²⁺ 2+ barium ion Zn²⁺ 2+zinc ion Cd²⁺ 2+ cadmium ion Al³⁺ 3+ aluminum ion Polyatomic Cations NH₄⁺ 1+ ammonium ion H₃O⁺ 1+ hydronium ion Multivalent Cations Cr²⁺ 2chromium(II) or chromous ion Cr³⁺ 3 chromium(III)or chromic ion Mn²⁺ 2manganese(II) or manganous ion Mn⁴⁺ 4 manganese(IV) ion Fe²⁺ 2 iron(II)or ferrous ion Fe³⁺ 3 iron(III) or ferric ion Co²⁺ 2 cobalt(II) orcobaltous ion Co³⁺ 3 cobalt(II) or cobaltic ion Ni²⁺ 2 nickel(II) ornickelous ion Ni³⁺ 3 nickel(III) or nickelic ion Cu⁺ 1 copper(I) orcuprous ion Cu²⁺ 2 copper(II) or cupric ion Sn²⁺ 2 tin(II) or atannousion Sn⁴⁺ 4 tin(IV) or atannic ion Pb²⁺ 2 lead(II) or plumbous ion Pb⁴⁺ 4lead(IV) or plumbic ion Monoatomic Anions H⁻ 1− hydride ion F⁻ 1−fluoride ion Cl⁻ 1− chloride ion Br⁻ 1− bromide ion I⁻ 1− iodide ion O²⁻2− oxide ion S²⁻ 2− sulfide ion N³⁻ 3− nitride ion Polyatomic Anions OH⁻1− hydroxide ion CN⁻ 1− cyanide ion SCN⁻ 1− thiocyanate ion C₂H₃O₂ ⁻ 1−acetate ion ClO⁻ 1− hypochlorite ion ClO₂ ⁻ 1− chlorite ion ClO₃ ⁻ 1−chlorate ion ClO₄ ⁻ 1− perchlorate ion NO₂ ⁻ 1− nitrite ion NO₃ ⁻ 1−nitrate ion MnO₄ ²⁻ 2− permanganate ion CO₃ ²⁻ 2− carbonate ion C₂O₄ ²⁻2− oxalate ion CrO₄ ²⁻ 2− chromate ion Cr₂O₇ ²⁻ 2− dichromate ion SO₃ ²⁻2− sulfite ion SO₄ ²⁻ 2− sulfate ion PO₃ ³⁻ 3− phosphite ion PO₄ ³⁻ 3−phosphate ion

The present disclosure sets forth novel gas-enriched fluids, including,but not limited to gas-enriched ionic aqueous solutions, aqueous salinesolutions (e.g., standard aqueous saline solutions, and other salinesolutions as discussed herein and as would be recognized in the art,including any physiological compatible saline solutions), cell culturemedia (e.g., minimal medium, and other culture media). A medium, ormedia, is termed “minimal” if it only contains the nutrients essentialfor growth. For prokaryotic host cells, a minimal media typicallyincludes a source of carbon, nitrogen, phosphorus, magnesium, and traceamounts of iron and calcium. (Gunsalus and Stanter, The Bacteria, V. 1,Ch. 1 Acad. Press Inc., N.Y. (1960)). Most minimal media use glucose asa carbon source, ammonia as a nitrogen source, and orthophosphate (e.g.,PO₄) as the phosphorus source. The media components can be varied orsupplemented according to the specific prokaryotic or eukaryoticorganism(s) grown, in order to encourage optimal growth withoutinhibiting target protein production. (Thompson et al., Biotech. andBioeng. 27: 818-824 (1985)).

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to modulate ¹³C-NMR line-widths of reporter solutes (e.g.,Trehelose) dissolved therein. NMR line-width effects are in indirectmethod of measuring, for example, solute ‘tumbling’ in a test fluid asdescribed herein in particular working Examples.

In particular aspects, the electrokinetically altered aqueous fluids arecharacterized by at least one of: distinctive square wave voltametrypeak differences at any one of −0.14V, −0.47V, −1.02V and −1.36V;polarographic peaks at −0.9 volts; and an absence of polarographic peaksat −0.19 and −0.3 volts, which are unique to the electrokineticallygenerated fluids as disclosed herein in particular working Examples.

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to alter cellular membrane conductivity (e.g., avoltage-dependent contribution of the whole-cell conductance as measurein patch clamp studies disclosed herein).

In particular aspects, the electrokinetically altered aqueous fluids aregasified (e.g., oxygenated), wherein the gas (e.g., oxygen) in the fluidis present in an amount of at least 15, ppm, at least 25 ppm, at least30 ppm, at least 40 ppm, at least 50 ppm, or at least 60 ppm dissolvedgas (e.g., oxygen) at atmospheric pressure. In particular aspects, theelectrokinetically altered aqueous fluids have less than 15 ppm, lessthat 10 ppm of dissolved gas (e.g., oxygen) at atmospheric pressure, orapproximately ambient oxygen levels.

In particular aspects, the electrokinetically altered aqueous fluids areoxygenated, wherein the gas (e.g., oxygen) in the fluid is present in anamount between approximately 8 ppm and approximately 15 ppm, and in thiscase is sometimes referred to herein as “Solas.”

In particular aspects, the electrokinetically altered aqueous fluidcomprises at least one of solvated electrons (e.g., stabilized bymolecular oxygen), and electrokinetically modified and/or charged gas(e.g., oxygen) species, and wherein in certain embodiments the solvatedelectrons and/or electrokinetically modified or charged gas (e.g.,oxygen) (species are present in an amount of at least 0.01 ppm, at least0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5ppm, at least 7 ppm, at least 10 ppm, at least 15 ppm, or at least 20ppm.

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to alter cellular membrane structure or function (e.g.,altering of a conformation, ligand binding activity, or a catalyticactivity of a membrane associated protein) sufficient to provide formodulation of intracellular signal transduction, wherein in particularaspects, the membrane associated protein comprises at least one selectedfrom the group consisting of receptors, transmembrane receptors (e.g.,G-Protein Coupled Receptor (GPCR), TSLP receptor, beta 2 adrenergicreceptor, bradykinin receptor, etc.), ion channel proteins,intracellular attachment proteins, cellular adhesion proteins, andintegrins. In certain aspects, the effected G-Protein Coupled Receptor(GPCR) interacts with a G protein α subunit (e.g., Gα_(s), Gα_(i),Gα_(q), and Gα₁₂).

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to modulate intracellular signal transduction, comprisingmodulation of a calcium dependant cellular messaging pathway or system(e.g., modulation of phospholipase C activity, or modulation ofadenylate cyclase (AC) activity).

In particular aspects, the electrokinetically altered aqueous fluids arecharacterized by various biological activities (e.g., regulation ofcytokines, receptors, enzymes and other proteins and intracellularsignaling pathways) described in the working Examples and elsewhereherein.

In particular aspects, the electrokinetically altered aqueous fluidsdisplay synergy with glatiramer acetate interferon-β, mitoxantrone,and/or natalizumab. In particular aspects, the electrokineticallyaltered aqueous fluids reduce DEP-induced TSLP receptor expression inbronchial epithelial cells (BEC) as shown in working Examples herein.

In particular aspects, the electrokinetically altered aqueous fluidsinhibit the DEP-induced cell surface-bound MMP9 levels in bronchialepithelial cells (BEC) as shown in working Examples herein.

In particular aspects, the biological effects of the electrokineticallyaltered aqueous fluids are inhibited by diphtheria toxin, indicatingthat beta blockade, GPCR blockade and Ca channel blockade affects theactivity of the electrokinetically altered aqueous fluids (e.g., onregulatory T cell function) as shown in working Examples herein.

In particular aspects, the physical and biological effects (e.g., theability to alter cellular membrane structure or function sufficient toprovide for modulation of intracellular signal transduction) of theelectrokinetically altered aqueous fluids persists for at least two, atleast three, at least four, at least five, at least 6 months, or longerperiods, in a closed container (e.g., closed gas-tight container).

Therefore, further aspects provide said electrokinetically-generatedsolutions and methods of producing an electrokinetically alteredgasofied (e.g., oxygenated) aqueous fluid or solution, comprising:providing a flow of a fluid material between two spaced surfaces inrelative motion and defining a mixing volume therebetween, wherein thedwell time of a single pass of the flowing fluid material within andthrough the mixing volume is greater than 0.06 seconds or greater than0.1 seconds; and introducing oxygen (O₂) into the flowing fluid materialwithin the mixing volume under conditions suitable to dissolve at least20 ppm, at least 25 ppm, at least 30, at least 40, at least 50, or atleast 60 ppm gas (e.g., oxygen) into the material, andelectrokinetically alter the fluid or solution. In certain aspects, thegas (e.g., oxygen) is infused into the material in less than 100milliseconds, less than 200 milliseconds, less than 300 milliseconds, orless than 400 milliseconds. In particular embodiments, the ratio ofsurface area to the volume is at least 12, at least 20, at least 30, atleast 40, or at least 50.

Yet further aspects, provide a method of producing an electrokineticallyaltered gasified (e.g., oxygenated) aqueous fluid or solution,comprising: providing a flow of a fluid material between two spacedsurfaces defining a mixing volume therebetween; and introducing gas(e.g., oxygen) into the flowing material within the mixing volume underconditions suitable to infuse at least 20 ppm, at least 25 ppm, at least30, at least 40, at least 50, or at least 60 ppm gas (e.g., oxygen) intothe material in less than 100 milliseconds, less than 200 milliseconds,less than 300 milliseconds, or less than 400 milliseconds. In certainaspects, the dwell time of the flowing material within the mixing volumeis greater than 0.06 seconds or greater than 0.1 seconds. In particularembodiments, the ratio of surface area to the volume is at least 12, atleast 20, at least 30, at least 40, or at least 50.

Additional embodiments provide a method of producing anelectrokinetically altered gasified (e.g., oxygenated) aqueous fluid orsolution, comprising use of a mixing device for creating an outputmixture by mixing a first material and a second material, the devicecomprising: a first chamber configured to receive the first materialfrom a source of the first material; a stator; a rotor having an axis ofrotation, the rotor being disposed inside the stator and configured torotate about the axis of rotation therein, at least one of the rotor andstator having a plurality of through-holes; a mixing chamber definedbetween the rotor and the stator, the mixing chamber being in fluidcommunication with the first chamber and configured to receive the firstmaterial therefrom, and the second material being provided to the mixingchamber via the plurality of through-holes formed in the one of therotor and stator; a second chamber in fluid communication with themixing chamber and configured to receive the output material therefrom;and a first internal pump housed inside the first chamber, the firstinternal pump being configured to pump the first material from the firstchamber into the mixing chamber. In certain aspects, the first internalpump is configured to impart a circumferential velocity into the firstmaterial before it enters the mixing chamber.

Further embodiments provide a method of producing an electrokineticallyaltered gasified (e.g., oxygenated) aqueous fluid or solution,comprising use of a mixing device for creating an output mixture bymixing a first material and a second material, the device comprising: astator; a rotor having an axis of rotation, the rotor being disposedinside the stator and configured to rotate about the axis of rotationtherein; a mixing chamber defined between the rotor and the stator, themixing chamber having an open first end through which the first materialenters the mixing chamber and an open second end through which theoutput material exits the mixing chamber, the second material enteringthe mixing chamber through at least one of the rotor and the stator; afirst chamber in communication with at least a majority portion of theopen first end of the mixing chamber; and a second chamber incommunication with the open second end of the mixing chamber.

Additional aspects provide an electrokinetically altered gasified (e.g.,oxygenated) aqueous fluid or solution made according to any of the abovemethods.

The term “treating” refers to, and includes, reversing, alleviating,inhibiting the progress of, or preventing a disease, disorder orcondition, or one or more symptoms thereof; and “treatment” and“therapeutically” refer to the act of treating, as defined herein.

A “therapeutically effective amount” is any amount of any of thecompounds utilized in the course of practicing the invention providedherein that is sufficient to reverse, alleviate, inhibit the progressof, or prevent a disease, disorder or condition, or one or more symptomsthereof.

Electrokinetically Gas (e.g., Oxygen)-Enriched Aqueous Fluids andSolutions

In the Figures and Examples that follow, it is to be understood thatwhile oxygen is the preferred gas used in producing the inventiveelectrokinetic fluids, and the describe biological activities, thepresent invention encompasses other electrokinetically-altered fluidscomprising the use of various positive ions and counter ions asdiscussed herein above. In certain aspects, such electrokinetic fluidvariants have the ability to modulate at least one of cellular membranepotential or cellular membrane conductance, and/or modulate at least onebiological activity as taught herein with the exemplary oxygenembodiments that comprise charge-stabilized oxygen-containingnanostructures substantially having an average diameter less than about100 nm.

FIG. 2 provides a block diagram illustrating some of the components of amixing device 100 and the flow of material into, within, and out of thedevice. The mixing device 100 combines two or more input materials toform an output material 102, which may be received therefrom into astorage vessel 104. The mixing device 100 agitates the two or more inputmaterials in a novel manner to produce an output material 102 havingnovel characteristics. The output material 102 may include not only asuspension of at least one of the input materials in at least one of theother input materials (e.g., emulsions) but also a novel combination(e.g., electrostatic combinations) of the input materials, a chemicalcompound resulting from chemical reactions between the input materials,combinations having novel electrostatic characteristics, andcombinations thereof.

The input materials may include a first material 110 provided by asource 112 of the first material, a second material 120 provided by asource 122 of the second material, and optionally a third material 130provided by a source 132 of the third material. The first material 110may include a liquid, such as water, saline solution, chemicalsuspensions, polar liquids, non-polar liquids, colloidal suspensions,cell growing media, and the like. In some embodiments, the firstmaterial 110 may include the output material 102 cycled back into themixing device 100. The second material 120 may consist of or include agas, such as oxygen, nitrogen, carbon dioxide, carbon monoxide, ozone,sulfur gas, nitrous oxide, nitric oxide, argon, helium, bromine, andcombinations thereof, and the like. In preferred embodiments, the gas isor comprises oxygen. The optional third material 130 may include eithera liquid or a gas. In some embodiments, the third material 130 may be orinclude the output material 102 cycled back into the mixing device 100(e.g., to one or more of the pumps 210, 220 or 230, and/or into thechamber 310, and/or 330).

Optionally, the first material 110, the second material 120, and theoptional third material 130 may be pumped into the mixing device 100 byan external pump 210, an external pump 220, and an external pump 230,respectively. Alternatively, one or more of the first material 110, thesecond material 120, and the optional third material 130 may be storedunder pressure in the source 112, the source 122, and the source 132,respectively, and may be forced into the mixing device 100 by thepressure. The invention is not limited by the method used to transferthe first material 110, the second material 120, and optionally, thethird material 130 into the mixing device 100 from the source 112, thesource 122, and the source 132, respectively.

The mixing device 100 includes a first chamber 310 and a second chamber320 flanking a mixing chamber 330. The three chambers 310, 320, and 330are interconnected and form a continuous volume.

The first material 110 is transferred into the first chamber 310 andflows therefrom into the mixing chamber 330. The first material 110 inthe first chamber 310 may be pumped into the first chamber 310 by aninternal pump 410. The second material 120 is transferred into themixing chamber 330. Optionally, the third material 130 may betransferred into the mixing chamber 330. The materials in the mixingchamber 330 are mixed therein to form the output material 102. Then, theoutput material 102 flows into the second chamber 320 from which theoutput material 102 exits the mixing device 100. The output material 102in the mixing chamber 330 may be pumped into the second chamber 320 byan internal pump 420. Optionally, the output material 102 in the secondchamber 320 may be pumped therefrom into the storage vessel 104 by anexternal pump 430 (e.g., alone or in combination with the internal pump410 and/or 420).

In particular aspects, a common drive shaft 500 powers both the internalpump 410 and the internal pump 420. The drive shaft 500 passes throughthe mixing chamber 330 and provides rotational force therein that isused to mix the first material 110, the second material 120, andoptionally, the third material 130 together. The drive shaft 500 ispowered by a motor 510 coupled thereto.

FIG. 3 provides a system 512 for supplying the first material 110 to themixing device 100 and removing the output material 102 from the mixingdevice 100. In the system 512, the storage vessel 104 of the outputmaterial 102 and the source 112 of the first material 110 are combined.The external pump 210 is coupled to the combined storage vessel 104 andsource 112 by a fluid conduit 514 such as hose, pipe, and the like. Theexternal pump 210 pumps the combined first material 110 and outputmaterial 102 from the combined storage vessel 104 and source 112 throughthe fluid conduit 514 and into a fluid conduit 516 connecting theexternal pump 210 to the mixing device 100. The output material 102exits the mixing device 100 through a fluid conduit 518. The fluidconduit 518 is coupled to the combined storage vessel 104 and source 112and transports the output material 102 exiting the mixing device 100 tothe combined storage vessel 104 and source 112. The fluid conduit 518includes a valve 519 that establishes an operating pressure or backpressure within the mixing device 100.

Referring to FIGS. 2, 4-10, and 11, a more detailed description ofvarious components of an embodiment of the mixing device 100 will beprovided. The mixing device 100 is scalable. Therefore, dimensionsprovided with respect to various components may be used to construct anembodiment of the device or may be scaled to construct a mixing deviceof a selected size.

Turning to FIG. 4, the mixing device 100 includes a housing 520 thathouses each of the first chamber 310, the mixing chamber 330, and thesecond chamber 320. As mentioned above, the mixing device 100 includesthe drive shaft 500, which rotates during operation of the device.Therefore, the mixing device 100 may vibrate or otherwise move.Optionally, the mixing device 100 may be coupled to a base 106, whichmay be affixed to a surface such as the floor to maintain the mixingdevice 100 in a substantially stationary position.

The housing 520 may be assembled from two or more housing sections. Byway of example, the housing 520 may include a central section 522flanked by a first mechanical seal housing 524 and a second mechanicalseal housing 526. A bearing housing 530 may be coupled to the firstmechanical seal housing 524 opposite the central section 522. A bearinghousing 532 may be coupled to the second mechanical seal housing 526opposite the central section 522. Optionally, a housing section 550 maybe coupled to the bearing housings 530.

Each of the bearing housings 530 and 532 may house a bearing assembly540 (see FIGS. 5 and 6). The bearing assembly 540 may include anysuitable bearing assembly known in the art including a model number“202SZZST” manufactured by SKF USA Inc, of Kulpsville, Pennsylvania,operating a website at www.skf.com.

Seals may be provided between adjacent housing sections. For example,o-ring 560 (see FIG. 5) may be disposed between the housing section 550and the bearing housing 530, o-ring 562 (see FIG. 5) may be disposedbetween the first mechanical seal housing 524 and the central section522, and o-ring 564 (see FIG. 6) may be disposed between the secondmechanical seal housing 526 and the central section 522.

Mixing Chamber 330

Turning now to FIG. 7, the mixing chamber 330 is disposed inside thecentral section 522 of the housing 520 between the first mechanical sealhousing 524 and the second mechanical seal housing 526. The mixingchamber 330 is formed between two components of the mixing device 100, arotor 600 and a stator 700. The rotor 600 may have a sidewall 604 withan inside surface 605 defining a generally hollow inside portion 610 andan outside surface 606. The sidewall 604 may be about 0.20 inches toabout 0.75 inches thick. In some embodiments, the sidewall 604 is about0.25 inches thick. However, because the mixing device 100 may be scaledto suit a particular application, embodiments of the device having asidewall 604 that is thicker or thinner than the values provided arewithin the scope of the present teachings. The sidewall 604 includes afirst end portion 612 and a second end portion 614 and a plurality ofthrough-holes 608 formed between the first end portion 612 and thesecond end portion 614. Optionally, the outside surface 606 of thesidewall 604 may include other features such as apertures, projections,textures, and the like. The first end portion 612 has a relieved portion616 configured to receive a collar 618 and the second end portion 614has a relieved portion 620 configured to receive a collar 622.

The rotor 600 is disposed inside the stator 700. The stator 700 has asidewall 704 with an inside surface 705 defining a generally hollowinside portion 710 into which the rotor 600 is disposed. The sidewall704 may be about 0.1 inches to about 0.3 inches thick. In someembodiments, the sidewall 604 is about 1.5 inches thick. The stator 700may be non-rotatably coupled to the housing 520 in a substantiallystationary position. Alternatively, the stator 700 may integrally formedwith the housing 520. The sidewall 704 has a first end portion 712 and asecond end portion 714. Optionally, a plurality of apertures 708 areformed in the sidewall 704 of the stator 700 between the first endportion 712 and the second end portion 714. Optionally, the insidesurface 705 of the sidewall 704 may include other features such asthrough-holes, projections, textures, and the like.

The rotor 600 rotates with respect to the stationary stator 700 about anaxis of rotation “α” in a direction indicated by arrow “C3” in FIG. 9.Each of the rotor 600 and the stator 700 may be generally cylindrical inshape and have a longitudinal axis. The rotor 600 has an outer diameter“D1” and the stator 700 may have an inner diameter “D2.” The diameter“D1” may range, for example, from about 0.5 inches to about 24 inches.In some embodiments, the diameter “D1” is about 3.04 inches. In someembodiments, the diameter “D1” is about 1.7 inches. The diameter “D2,”which is larger than the diameter “D1,” may range from about 0.56 inchesto about 24.25 inches. In some embodiments, the diameter “D2” is about 4inches. Therefore, the mixing chamber 330 may have a ring-shapedcross-sectional shape that is about 0.02 inches to about 0.125 inchesthick (i.e., the difference between the diameter “D2” and the diameter“D1”). In particular embodiments, the mixing chamber 330 is about 0.025inches thick. The channel 32 between the rotor 12 and the stator 34 ofprior art device 10 (see FIG. 1) has a ring-shaped cross-sectional shapethat is about 0.09 inches thick. Therefore, in particular embodiments,the thickness of the mixing chamber 330 is less than about one third ofthe channel 32 of the prior art device 10.

The longitudinal axis of the rotor 600 may be aligned with its axis ofrotation “α.” The longitudinal axis of the rotor 600 may be aligned withthe longitudinal axis of the stator 700. The rotor 600 may have a lengthof about 3 inches to about 6 inches along the axis of rotation “α.” Insome embodiments, the rotor 600 may have a length of about inches alongthe axis of rotation “α.” The stator 700 may have a length of about 3inches to about 6 inches along the axis of rotation “α.” In someembodiments, the stator 700 may have a length of about 5 inches alongthe axis of rotation “α.”

While the rotor 600 and the stator 700 have been depicted as having agenerally cylindrical shape, those of ordinary skill in the artappreciate that alternate shapes may be used. For example, the rotor 600and the stator 700 may be conically, spherically, arbitrarily shaped,and the like. Further, the rotor 600 and the stator 700 need not beidentically shaped. For example, the rotor 600 may be cylindricallyshaped and the stator 700 rectangular shaped or vise versa.

The apertures 708 of the stator 700 and the through-holes 608 depictedin FIGS. 4-7 are generally cylindrically shaped. The diameter of thethrough-holes 608 may range from about 0.1 inches to about 0.625 inches.The diameter of the apertures 708 may range from about 0.1 inches toabout 0.625 inches. One or more of apertures 708 of the stator 700 mayhave a diameter that differs from the diameters of the other apertures708. For example, the apertures 708 may increase in diameter from thefirst end portion 712 of the stator 700 to the second end portion 714 ofthe stator 700, the apertures 708 may decrease in diameter from thefirst end portion 712 of the stator 700 to the second end portion 714 ofthe stator 700, or the diameters of the apertures 708 may vary inanother manner along the stator 700. One or more of through-holes 608 ofthe rotor 600 may have a diameter that differs from the diameters of theother through-holes 608. For example, the through-holes 608 may increasein diameter from the first end portion 612 of the rotor 600 to thesecond end portion 614 of the rotor 600, the through-holes 608 maydecrease in diameter from the first end portion 612 of the rotor 600 tothe second end portion 614 of the rotor 600, or the diameters of thethrough-holes 608 may vary in another manner along the rotor 600.

As described below with reference to alternate embodiments, theapertures 708 and the through-holes 608 may have shapes other thangenerally cylindrical and such embodiments are within the scope of thepresent invention. For example, the through-holes 608 may include anarrower portion, an arcuate portion, a tapered portion, and the like.Referring to FIG. 7, each of the through-holes 608 includes an outerportion 608A, a narrow portion 608B, and a tapered portion 608Cproviding a transition between the outer portion 608A and the narrowportion 608B. Similarly, the apertures 708 may include a narrowerportion, an arcuate portion, a tapered portion, and the like.

FIG. 8 provides a non-limiting example of a suitable arrangement of theapertures 708 of the stator 700 and the through-holes 608 of the rotor600. The apertures 708 of the stator 700 may be arranged insubstantially parallel lateral rows “SLAT-1” through “SLAT-6”substantially orthogonal to the axis of rotation “α.” The apertures 708of the stator 700 may also be arranged in substantially parallellongitudinal rows “SLONG-1” through “SLONG-7” substantially parallelwith the axis of rotation “α.” In other words, the apertures 708 of thestator 700 may be arranged in a grid-like pattern of orthogonal rows(i.e., the lateral rows are orthogonal to the longitudinal rows) havingthe longitudinal rows “SLONG-1” through “SLONG-7” substantially parallelwith the axis of rotation “α.”

Like the apertures 708 of the stator 700, the through-holes 608 of therotor 600 may be arranged in substantially parallel lateral rows“RLAT-1” through “RLAT-6” substantially orthogonal to the axis ofrotation “α.” However, instead of being arranged in a grid-like patternof orthogonal rows, the through-holes 608 of the rotor 600 may also bearranged in substantially parallel rows “RLONG-1” through “RLONG-7” thatextend longitudinally along a helically path. Alternatively, thethrough-holes 608 of the rotor 600 may also be arranged in substantiallyparallel rows “RLONG-1” through “RLONG-7” that extend longitudinally atan angle other than parallel with the axis of rotation “α.”

The apertures 708 of the stator 700 and the through-holes 608 of therotor 600 may be configured so that when the rotor 600 is disposedinside the stator 700 the lateral rows “SLAT-1” to “SLAT-6” at leastpartially align with the lateral rows “RLAT-1” to “RLAT-6,”respectively. In this manner, as the rotor 600 rotates inside the stator700, the through-holes 608 pass by the apertures 708.

The through-holes 608 in each of the lateral rows “RLAT-1” to “RLAT-6”may be spaced apart laterally such that all of the through-holes 608 inthe lateral row align, at least partially, with the apertures 708 in acorresponding one of the lateral rows “SLAT-1” to “SLAT-6” of the stator700 at the same time. The longitudinally extending rows “RLONG-1”through “RLONG-6” may be configured such that the through-holes 608 inthe first lateral row “RLAT-1” in each of the longitudinally extendingrows passes completely by the apertures 708 of the corresponding lateralrow “SLAT-1” before the through-holes 608 in the last lateral row“RLAT-6” begin to partially align with the apertures 708 of thecorresponding last lateral row “SLAT-6” of the stator 700.

While, in FIG. 8, six lateral rows and six longitudinally extending rowshave been illustrated with respect to the rotor 600 and six lateral rowsand seven longitudinally extending rows have been illustrated withrespect stator 700, it is apparent to those of ordinary skill in the artthat alternate numbers of lateral rows and/or longitudinal rows may beused with respect to the rotor 600 and/or stator 700 without departingfrom the present teachings.

To ensure that only one pair of openings between corresponding lateralrows will be coincident at any one time, the number of apertures 708 ineach of the lateral rows “SLAT-1” to “SLAT-6” on the stator 700 maydiffer by a predetermined number (e.g., one, two, and the like) thenumber of through-holes 608 in each of the corresponding lateral rows“RLAT-1” to “RLAT-6” on the rotor 600. Thus, for example, if lateral row“RLAT-1” has twenty through-holes 608 evenly spaced around thecircumference of rotor 600, the lateral row “SLAT-1” may have twentyapertures 708 evenly spaced around the circumference of stator 700.

Returning to FIG. 7, the mixing chamber 330 has an open first endportion 332 and an open second end portion 334. The through-holes 608formed in the sidewall 604 of the rotor 600 connect the inside portion610 of the rotor 600 with the mixing chamber 330.

The rotor 600 is rotated inside the stator 700 by the drive shaft 500aligned with the axis of rotation “α” of the rotor 600. The drive shaft500 may be coupled to the first end portion 612 and the second endportion 614 of the rotor 600 and extend through its hollow insideportion 610. In other words, a portion 720 of the drive shaft 500 isdisposed in the hollow inside portion 610 of the rotor 600.

The collar 618 is configured to receive a portion 721 of the drive shaft500 disposed in the hollow inside portion 610 and the collar 622 isconfigured to receive a portion 722 of the drive shaft 500 disposed inthe hollow inside portion 610.

The portion 721 has an outer diameter “D3” that may range from about 0.5inches to about 2.5 inches. In some embodiments, the diameter “D3” isabout 0.625 inches. The portion 722 has an outer diameter “D4” that maybe substantially similar to the diameter “D3,” although, this is notrequired. The diameter “D4” may range from about 0.375 inches to about2.5 inches.

The rotor 600 may be non-rotationally affixed to the portion 721 and theportion 722 of the drive shaft 500 by the collar 618 and the collar 622,respectively. By way of example, each of the collars 618 and 622 may beinstalled inside relieved portions 616 and 620, respectively. Then, thecombined rotor 600 and collars 618 and 622 may be heated to expand them.Next, the drive shaft 500 is inserted through the collars 618 and 622and the assembly is allowed to cool. As the collars 618 and 622 shrinkduring cooling, they tighten around the portions 722A and 722B of thedrive shaft 500, respectively, gripping it sufficiently tightly toprevent the drive shaft 500 from rotating relative to the rotor 600. Thecollar 618, which does not rotate with respect to either the portion 721or the relieved portion 616, translates the rotation of the drive shaft500 to the first end portion 612 the rotor 600. The collar 622, whichdoes not rotate with respect to either the portion 722 or the relievedportion 620, translates the rotation of the drive shaft 500 to thesecond end portion 614 of the rotor 600. The drive shaft 500 and therotor 600 rotate together as a single unit.

The drive shaft 500 may have a first end portion 724 (see FIG. 5) and asecond end portion 726 (see FIG. 6). The first end portion 724 may havea diameter “D5” of about 0.5 inches to about 1.75 inches. In particularembodiments, the diameter “D5” may be about 1.25 inches. The second endportion 726 may have a diameter “D6” that may be substantially similarto diameter “D5.”

The second material 120 may be transported into the mixing chamber 330through one of the first end portion 724 and the second end portion 726of the rotating drive shaft 500. The other of the first end portion 724and the second end portion 726 of the drive shaft 500 may be coupled tothe motor 510. In the embodiment depicted in FIGS. 5 and 6, the secondmaterial 120 is transported into the mixing chamber 330 through thefirst end portion 724 and the second end portion 726 of the drive shaft500 is coupled to the motor 510.

Turning to FIG. 5, the drive shaft 500 may have a channel 728 formedtherein that extends from first end portion 724 into the portion 720disposed in the inside portion 610 of the rotor 600. The channel 728 hasan opening 730 formed in the first end portion 724. When the mixingdevice 100 is operating, the second material 120 is introduced into thechannel 728 through the opening 730.

A valve 732 may be disposed inside a portion of the channel 728 locatedin the first end portion 724 of the drive shaft 500. The valve 732 mayrestrict or otherwise control the backward flow of the second material120 from inside the hollow inside portion 610 through the channel 728and/or the forward flow of the second material 120 into the channel 728.The valve 732 may include any valve known in the art including a checkvalve. A suitable check valve includes a part number “CKFA1876205A,”free flow forward check valve, manufactured by The Lee Company USAhaving an office in Bothell, Wash. and operating a website atwww.theleeco.com.

The drive shaft 500 may include an aperture 740 located in the insideportion 610 of the rotor 600 that connects the channel 728 with theinside portion 610 of the rotor 600. While only a single aperture 740 isillustrated in FIG. 5, it is apparent to those of ordinary skill in theart that multiple apertures may be used to connect the channel 728 withthe inside portion 610 of the rotor 600.

Referring to FIG. 2, optionally, the external pump 220 may pump thesecond material 120 into the mixing device 100. The pump 220 may includeany suitable pump known in the art. By way of non-limiting example, thepump 220 may include any suitable pump known in the art including adiaphragm pump, a chemical pump, a peristaltic pump, a gravity fed pump,a piston pump, a gear pump, a combination of any of the aforementionedpumps, and the like. If the second material 120 is a gas, the gas may bepressurized and forced into the opening 730 formed in the first endportion 724 of the drive shaft 500 by releasing the gas from the source122.

The pump 220 or the source 122 is coupled to the channel 728 by thevalve 732. The second material 120 transported inside the channel 728exits the channel 728 into the inside portion 610 of the rotor 600through the aperture 740. The second material 120 subsequently exits theinside portion 610 of the rotor 600 through the through-holes 608 formedin the sidewall 608 of the rotor 600.

Referring to FIG. 5, the mixing device 100 may include a seal assembly750 coupled to the first end portion 724 of the drive shaft 500. Theseal assembly 750 is maintained within a chamber 752 defined in thehousing 520. The chamber 752 has a first end portion 754 spaced acrossthe chamber from a second end portion 756. The chamber 752 also includesan input port 758 and an output port 759 that provide access into thechamber 752. The chamber 752 may be defined by housing section 550 andthe bearing housing 530. The first end portion 754 may be formed in thehousing section 550 and the second end portion 756 may be adjacent tothe bearing housing 530. The input port 758 may be formed in the bearinghousing 530 and the output port 759 may be formed in the housing section550.

The seal assembly 750 includes a first stationary seal 760 installed inthe first end portion 754 of the chamber 752 in the housing section 550and the bearing housing 530. The first stationary seal 760 extendsaround a portion 762 of the first end portion 724 of the drive shaft500. The seal assembly 750 also includes a second stationary seal 766installed in the second end portion 756 of the chamber 752 in thebearing housing 530. The second stationary seal 766 extends around aportion 768 of the first end portion 724 of the drive shaft 500.

The seal assembly 750 includes a rotating assembly 770 that isnon-rotatably coupled to the first end portion 724 of the drive shaft500 between the portion 762 and the portion 768. The rotating assembly770 rotates therewith as a unit. The rotating assembly 770 includes afirst seal 772 opposite a second seal 774. A biasing member 776 (e.g., aspring) is located between the first seal 772 and the second seal 774.The biasing member 776 biases the first seal 772 against the firststationary seal 760 and biases the second seal 774 against the secondstationary seal 766.

A cooling lubricant is supplied to the chamber 752 and around rotatingassembly 770. The lubricant enters the chamber 752 through the inputport 758 and exits the chamber 752 through output port 759. Thelubricant may lubricate the bearing assembly 540 housed by the bearinghousing 530. A chamber 570 may be disposed between the bearing housing530 and the mechanical seal housing 524. The bearing housing 530 mayalso include a second input port 759 connected to the chamber 570 intowhich lubricant may be pumped. Lubricant pumped into the chamber 570 maylubricate the bearing assembly 540. The seal assembly 750 maysignificantly, if not greatly, reduce frictional forces within thisportion of the device caused by the rotation of the rotor 600 and mayincrease the active life of the seals 770. The seals may includesurfaces constructed using silicon carbide.

Referring to FIG. 9, as the rotor 600 rotates about the axis of rotation“α” in the direction indicated by arrow “C1,” the rotor expels thesecond material 120 into the mixing chamber 330. The expelled bubbles,droplets, particles, and the like of the second material 120 exit therotor 600 and are imparted with a circumferential velocity (in adirection indicated by arrow “C3”) by the rotor 600. The second material120 may forced from the mixing chamber 330 by the pump 220 (see FIG. 2),the centrifugal force of the rotating rotor 600, buoyancy of the secondmaterial 120 relative to the first material 110, and a combinationthereof.

Motor 510

Returning to FIG. 6, the second end portion 726 of the drive shaft 500may be coupled to a rotating spindle 780 of a motor 510 by a coupler900. The spindle 780 may have a generally circular cross-sectional shapewith a diameter “D7” of about 0.25 inches to about 2.5 inches. Inparticular embodiments, the diameter “D7” may be about 0.25 inches toabout 1.5 inches. While in the embodiment depicted in FIG. 6, thediameter “D5” of the first end portion 724 of the drive shaft 500 issubstantially equal to the diameter “D7” and the spindle 780,embodiments in which one of the diameter “D5” and the diameter “D7” islarger than the other are within the scope of the present invention.

Referring also to FIG. 4, it may be desirable to cover or shield thecoupler 900. In the embodiment illustrated in FIGS. 4 and 6, a driveguard 910 covers the coupler 900. The drive guard 910 may be generallyU-shaped having a curved portion 914 flanked by a pair of substantiallylinear portions 915 and 916. The distal end of each of the substantiallylinear portions 915 and 916 of the drive guard 910 may have a flange 918and 919, respectively. The drive guard 910 may be fastened by each ofits flanges 918 and 919 to the base 106.

The motor 510 may be supported on the base 106 by a support member 920.The support member 920 may be coupled to the motor 510 near the spindle780. In the embodiment depicted, the support member 920 includes athrough-hole through which the spindle 780 passes. The support member920 may be coupled to the motor 510 using any method known in the art,including bolting the support member 920 to the motor 510 with one ormore bolts 940.

The coupler 900 may include any coupler suitable for transmitting asufficient amount of torque from the spindle 780 to the drive shaft 500to rotate the rotor 600 inside to the stator 700. In the embodimentillustrated in FIGS. 4 and 6, the coupler 900 is a bellows coupler. Abellows coupler may be beneficial if the spindle 780 and the drive shaft500 are misaligned. Further, the bellows coupler may help absorb axialforces exerted on the drive shaft 500 that would otherwise be translatedto the spindle 780. A suitable bellows coupler includes a model“BC32-8-8-A,” manufactured by Ruland Manufacturing Company, Inc. ofMarlborough, Mass., which operates a website at www.ruland.com.

The motor 510 may rotate the rotor 600 at about 0.1 revolutions perminute (“rpm”) to about 7200 rpm. The motor 510 may include any motorsuitable for rotating the rotor 600 inside to the stator 700 inaccordance with the present teachings. By way of non-limiting example, asuitable motor may include a one-half horsepower electric motor,operating at 230/460 volts and 3450 per minute (“rpm”). A suitable motorincludes a model “C4T34NC4C” manufactured by LEESON Electric Corporationof Grafton, Wis., which operates a website at www.leeson.com.

First Chamber 310

Turning to FIGS. 4 and 7, the first chamber 320 is disposed inside thecentral section 522 of the housing 520 between the first mechanical sealhousing 524 and the first end portions 612 and 712 of the rotor 600 andthe stator 700, respectively. The first chamber 310 may be annular andhave a substantially circular cross-sectional shape. The first chamber310 and the mixing chamber 330 form a continuous volume. A portion 1020of the drive shaft 500 extends through the first chamber 310.

As may best be viewed in FIG. 4, the first chamber 310 has an input port1010 through which the first material 110 enters the mixing device 100.The first material 110 may be pumped inside the first chamber 310 by theexternal pump 210 (see FIG. 2). The external pump 210 may include anypump known in the art for pumping the first material 110 at a sufficientrate to supply the first chamber 310.

The input port 1010 is oriented substantially orthogonally to the axisof rotation “α.” Therefore, the first material 110 enters the firstchamber 310 with a velocity tangential to the portion 1020 of the driveshaft 500 extending through the first chamber 310. The tangentialdirection of the flow of the first material 110 entering the firstchamber 310 is identified by arrow “T1.” In the embodiment depicted inFIGS. 4 and 7, the input port 1010 may be offset from the axis ofrotation “α.” As is apparent to those of ordinary skill in the art, thedirection of the rotation of the drive shaft 500 (identified by arrow“C1” in FIG. 9), has a tangential component. The input port 1010 ispositioned so that the first material 110 enters the first chamber 310traveling in substantially the same direction as the tangentialcomponent of the direction of rotation of the drive shaft 500.

The first material 110 enters the first chamber 310 and is deflected bythe inside of the first chamber 310 about the portion 1020 of the driveshaft 500. In embodiments wherein the first chamber 310 has asubstantially circular cross-sectional shape, the inside of the firstchamber 310 may deflect the first material 110 in a substantiallycircular path (identified by arrow “C2” in FIG. 9) about the portion1020 of the drive shaft 500. In such an embodiment, the tangentialvelocity of the first material 110 may cause it to travel about the axisof rotation “α” at a circumferential velocity, determined at least inpart by the tangential velocity.

Once inside the first chamber 310, the first material 110 may be pumpedfrom the first chamber 310 into the mixing chamber 330 by the pump 410residing inside the first chamber 310. In embodiments that include theexternal pump 210 (see FIG. 2), the external pump 210 may be configuredto pump the first material 110 into the first chamber 310 at a rate atleast as high as a rate at which the pump 410 pumps the first material110 from the first chamber 310.

The first chamber 310 is in communication with the open first endportion 332 of the mixing chamber 330 and the first material 110 insidethe first chamber 310 may flow freely into the open first end portion332 of the mixing chamber 330. In this manner, the first material 110does not negotiate any corners or bends between the mixing chamber 330and the first chamber 310. In the embodiment depicted, the first chamber310 is in communication with the entire open first end portion 332 ofthe mixing chamber 330. The first chamber 310 may be filled completelywith the first material 110.

The pump 410 is powered by the portion 1020 of the drive shaft 500extending through the first chamber 310. The pump 410 may include anypump known in the art having a rotating pump member 2022 housed inside achamber (i.e., the first chamber 310) defined by a stationary housing(i.e., the housing 520). Non-limiting examples of suitable pumps includerotary positive displacement pumps such as progressive cavity pumps,single screw pumps (e.g., Archimedes screw pump), and the like.

The pump 410 depicted in FIGS. 7 and 9, is generally referred to as asingle screw pump. In this embodiment, the pump member 2022 includes acollar portion 2030 disposed around the portion 1020 of the drive shaft500. The collar portion 2030 rotates with the portion 1020 of the driveshaft 500 as a unit. The collar portion 2030 includes one or more fluiddisplacement members 2040. In the embodiment depicted in FIGS. 7 and 9,the collar portion 2030 includes a single fluid displacement member 2040having a helical shape that circumscribes the collar portion 2030 alonga helical path.

Referring to FIG. 9, the inside of the first chamber 310 is illustrated.The pump 410 imparts an axial flow (identified by arrow “A1” and arrow“A2”) in the first material 110 inside the first chamber 310 toward theopen first end portion 332 of the mixing chamber 330. The axial flow ofthe first material 110 imparted by the pump 410 has a pressure that mayexceed the pressure obtainable by the external pump of the prior artdevice 10 (see FIG. 1).

The pump 410 may also be configured to impart a circumferential flow(identified by arrow “C2”) in the first material 110 as it travelstoward the open first end portion 332 of the mixing chamber 330. Thecircumferential flow imparted in the first material 110 before it entersthe mixing chamber 330 causes the first material 110 to enter the mixingchamber 330 already traveling in the desired direction at an initialcircumferential velocity. In the prior art device 10 depicted in FIG. 1,the first material 110 entered the channel 32 of the prior art device 10without a circumferential velocity. Therefore, the rotor 12 of the priorart device 10 alone had to impart a circumferential flow into the firstmaterial 110. Because the first material 110 is moving axially, in theprior art device 10, the first material 110 traversed at least a portionof the channel 32 formed between the rotor 12 and the stator 30 at aslower circumferential velocity than the first material 110 traversesthe mixing chamber 330 of the mixing device 100. In other words, if theaxial velocity of the first material 110 is the same in both the priorart device 10 and the mixing device 100, the first material 110 maycomplete more revolutions around the rotational axis “α” beforetraversing the axial length of the mixing chamber 330, than it wouldcomplete before traversing the axial length of the channel 32. Theadditional revolutions expose the first material 110 (and combined firstmaterial 110 and second material 120) to a substantially larger portionof the effective inside surface 706 (see FIG. 7) of the stator 700.

In embodiments including the external pump 210 (see FIG. 2), thecircumferential velocity imparted by the external pump 210 combined withthe input port 1010 being oriented according to the present teachings,may alone sufficiently increase the revolutions of the first material110 (and combined first material 110 and second material 120) about therotational axis “α.” Further, in some embodiments, the circumferentialvelocity imparted by the pump 210 and the circumferential velocityimparted by the pump 410 combine to achieve a sufficient number ofrevolutions of the first material 110 (and combined first material 110and second material 120) about the rotational axis “α.” As isappreciated by those of ordinary skill in the art, other structuralelements such as the cross-sectional shape of the first chamber 310 maycontribute to the circumferential velocity imparted by the pump 210, thepump 410, and a combination thereof.

In an alternate embodiment depicted in FIG. 10, the pump 410 may includeone or more vanes 2042 configured to impart a circumferential flow inthe first material 110 as it travels toward the open first end portion332 of the mixing chamber 330.

Second Chamber 320

Turning now to FIGS. 4 and 7, the second chamber 320 is disposed insidethe central section 522 of the housing 520 between the second mechanicalseal housing 526 and the second end portions 614 and 714 of the rotor600 and the stator 700, respectively. The second chamber 320 may besubstantially similar to the first chamber 310. However, instead of theinput port 1010, the second chamber 320 may include an output port 3010.A portion 3020 of the drive shaft 500 extends through the second chamber320.

The second chamber 320 and the mixing chamber 330 form a continuousvolume. Further, the first chamber 310, the mixing chamber 330, and thesecond chamber 320 form a continuous volume. The first material 110flows through the mixing device 100 from the first chamber 310 to themixing chamber 330 and finally to the second chamber 320. While in themixing chamber 330, the first material 110 is mixed with the secondmaterial 120 to form the output material 102. The output material 102exits the mixing device 100 through the output port 3010. Optionally,the output material 102 may be returned to the input port 1010 and mixedwith an additional quantity of the second material 120, the thirdmaterial 130, or a combination thereof.

The output port 3010 is oriented substantially orthogonally to the axisof rotation “α” and may be located opposite the input port 1010 formedin the first chamber 310. The output material 102 enters the secondchamber 320 from the mixing chamber 330 having a circumferentialvelocity (in the direction indicated by arrow “C3” in FIG. 9) impartedthereto by the rotor 600. The circumferential velocity is tangential tothe portion 3020 of the drive shaft 500 extending through the secondchamber 320. In the embodiment depicted in FIGS. 4, 6, and 7, the outputport 3010 may be offset from the axis of rotation “α.” The output port3010 is positioned so that the output material 102, which enters thesecond chamber 320 traveling in substantially the same direction inwhich the drive shaft 500 is rotating (identified in FIG. 9 by arrow“C1”), is traveling toward the output port 3010.

The output material 102 enters the second chamber 320 and is deflectedby the inside of the second chamber 320 about the portion 3020 of thedrive shaft 500. In embodiments wherein the second chamber 320 has asubstantially circular cross-sectional shape, the inside of the secondchamber 320 may deflect the output material 102 in a substantiallycircular path about the portion 3020 of the drive shaft 500.

Referring to FIG. 2, optionally, the output material 102 may be pumpedfrom inside the second chamber 320 by the external pump 430. Theexternal pump 430 may include any pump known in the art for pumping theoutput material 102 at a sufficient rate to avoid limiting throughput ofthe mixing device 100. In such an embodiment, the external pump 430 mayintroduce a tangential velocity (in a direction indicated by arrow “T2”in FIGS. 4 and 11) to at least a portion of the output material 102 asthe external pump 430 pumps the output material 102 from the secondchamber 320. The tangential velocity of the portion of the outputmaterial 102 may cause it to travel about the axis of rotation “α” at acircumferential velocity, determined in part by the tangential velocity.

Pump 420

Turning to FIGS. 6 and 7, the pump 420 residing inside the secondchamber 320 may pump the output material 102 from the second chamber 320into the output port 3010 and/or from the mixing chamber 330 into thesecond chamber 320. In embodiments that include the external pump 430,the external pump 430 may be configured to pump the output material 102from the second chamber 320 at a rate at least as high as a rate atwhich the pump 420 pumps the output material 102 into the output port3010.

The second chamber 320 is in communication with the open second endportion 334 of the mixing chamber 330 and the output material 102 insidethe mixing chamber 330 may flow freely from the open second end portion334 into the second chamber 320. In this manner, the output material 102does not negotiate any corners or bends between the mixing chamber 330and the second chamber 320. In the embodiment depicted, the secondchamber 320 is in communication with the entire open second end portion334 of the mixing chamber 330. The second chamber 320 may be filledcompletely with the output material 102.

The pump 420 is powered by the portion 3020 of the drive shaft 500extending through the second chamber 320. The pump 420 may besubstantially identical to the pump 410. Any pump described above assuitable for use as the pump 410 may be used for the pump 420. While thepump 410 pumps the first material 110 into the mixing chamber 330, thepump 420 pumps the output material 102 from the mixing chamber 330.Therefore, both the pump 410 and the pump 420 may be oriented to pump inthe same direction.

As is appreciated by those of ordinary skill in the art, the firstmaterial 110 may differ from the output material 102. For example, oneof the first material 110 and the output material 102 may be moreviscous than the other. Therefore, the pump 410 may differ from the pump420. The pump 410 may be configured to accommodate the properties of thefirst material 110 and the pump 420 may be configured to accommodate theproperties of the output material 102.

The pump 420 depicted in FIGS. 6 and 7, is generally referred to as asingle screw pump. In this embodiment, the pump member 4022 includes acollar portion 4030 disposed around the portion 3020 of the drive shaft500. The collar portion 4030 rotates with the portion 3020 of the driveshaft 500 as a unit. The collar portion 4030 includes one or more fluiddisplacement members 4040. The collar portion 4030 includes a singlefluid displacement member 4040 having a helical shape that circumscribesthe collar portion 4030 along a helical path.

Referring to FIG. 11, the inside of the second chamber 320 isillustrated. The pump 420 imparts an axial flow (identified by arrow“A3” and arrow “A4”) in the output material 102 inside the secondchamber 320 away from the open second end portion 334 of the mixingchamber 330.

The pump 420 may be configured to impart a circumferential flow(identified by arrow “C4”) in the output material 102 as it travels awayfrom the open second end portion 334 of the mixing chamber 330. Thecircumferential flow imparted in the output material 102 may help reducean amount of work required by the rotor 600. The circumferential flowalso directs the output material 102 toward the output port 3010.

In an alternate embodiment, the pump 420 may have substantially the sameconfiguration of the pump 410 depicted in FIG. 10. In such anembodiment, the one or more vanes 2042 are configured to impart acircumferential flow in the output material 102 as it travels away fromthe open second end portion 334 of the mixing chamber 330.

As is apparent to those of ordinary skill, various parameters of themixing device 100 may be modified to obtain different mixingcharacteristics. Exemplary parameters that may be modified include thesize of the through-holes 608, the shape of the through-holes 608, thearrangement of the through-holes 608, the number of through-holes 608,the size of the apertures 708, the shape of the apertures 708, thearrangement of the apertures 708, the number of apertures 708, the shapeof the rotor 600, the shape of the stator 700, the width of the mixingchamber 330, the length of the mixing chamber 330, rotational speed ofthe drive shaft 500, the axial velocity imparted by the internal pump410, the circumferential velocity imparted by the internal pump 410, theaxial velocity imparted by the internal pump 420, the circumferentialvelocity imparted by the internal pump 420, the configuration ofdisturbances (e.g., texture, projections, recesses, apertures, and thelike) formed on the outside surface 606 of the rotor 600, theconfiguration of disturbances (e.g., texture, projections, recesses,apertures, and the like) formed on the inside surface 706 of the stator700, and the like.

Alternate Embodiment

Referring to FIG. 12, a mixing device 5000 is depicted. The mixingdevice 5000 is an alternate embodiment of the mixing device 100.Identical reference numerals have been used herein to identifycomponents of the mixing device 5000 that are substantially similarcorresponding components of the mixing device 100. Only components ofthe mixing device 5000 that differ from the components of the mixingdevice 100 will be described.

The mixing device 5000 includes a housing 5500 for housing the rotor 600and the stator 5700. The stator 5700 may be non-rotatably couple by itsfirst end portion 5712 and its second end portion 5714 to the housing5500. A chamber 5800 is defined between the housing 5500 and a portion5820 of the stator 5700 flanked by the first end portion 5712 and thesecond end portion 5714. The housing 5500 includes an input port 5830which provides access into the chamber 5800. The input port 5830 may beoriented substantially orthogonally to the axis of rotation “α.”however, this is not a requirement.

The stator 5700 includes a plurality of through-holes 5708 that connectthe chamber 5800 and the mixing chamber 330 (defined between the rotor600 and the stator 5700). An external pump 230 may be used to pump thethird material 130 (which may be identical to the second material 120)into the chamber 5800 via the input port 5830. The third material 130pumped into the chamber 5800 may enter the mixing chamber 330 via thethrough-holes 5708 formed in the stator 5700. The third material 130 mayforced from the channel 5800 by the pump 230, buoyancy of the thirdmaterial 130 relative to the first material 110, and a combinationthereof. As the rotor 600 rotates, it may also draw the third material130 from the channel 5800 into the mixing chamber 330. The thirdmaterial 130 may enter the mixing chamber 330 as bubbles, droplets,particles, and the like, which are imparted with a circumferentialvelocity by the rotor 600.

Alternate Embodiment

An alternate embodiment of the mixing device 100 may be constructedusing a central section 5900 depicted in FIG. 13 and a bearing housing5920 depicted in FIG. 14. FIG. 13 depicts the central section 5900having in its interior the stator 700 (see FIG. 7). Identical referencenumerals have been used herein to identify components associated withthe central section 5900 that are substantially similar correspondingcomponents of the mixing device 100. Only components of the centralsection 5900 that differ from the components of the central section 522will be described. The central section 5900 and the stator 700 are bothconstructed from a conductive material such as a metal (e.g., stainlesssteel). The input port 1010 and the output port 3010 are bothconstructed from a nonconductive material such as plastic (e.g., PET,Teflon, nylon, PVC, polycarbonate, ABS, Delrin, polysulfone, etc.).

An electrical contact 5910 is coupled to the central section 5900 andconfigured to deliver a charge thereto. The central section 5900conducts an electrical charge applied to the electrical contact 5910 tothe stator 700. In further embodiments, the central section 5900 may beconstructed from a nonconductive material. In such embodiments, theelectrical contact 5910 may pass through the central section 5900 andcoupled to the stator 700. The electric charge applied by the electricalcontact 5910 to the stator 700 may help facilitate redox or otherchemical reactions inside the mixing chamber 330.

Optionally, insulation (not shown) may be disposed around the centralsection 5900 to electrically isolate it from the environment. Further,insulation may be used between the central section 5900 and the firstand second mechanical seals 524 and 526 that flank it to isolate itelectrically from the other components of the mixing device.

Turning now to FIG. 14, the bearing housing 5920 will be described. Thebearing housing 5920 is disposed circumferentially around the portion726 of the drive shaft 500. An electrical contact 5922 is coupled to thebearing housing 5920. A rotating brush contact 5924 provides anelectrical connection between the drive shaft 500 and the electricalcontact 5922.

In this embodiment, the drive shaft 500 and the rotor 600 are bothconstructed from a conductive material such as a metal (e.g., stainlesssteel). The bearing housing 5920 may be constructed from either aconductive or a nonconductive material. An electrical charge is appliedto the drive shaft 500 by the electrical contact 5922 and the rotatingbrush contact 5924. The electrical charge is conducted by the driveshaft 500 to the rotor 600.

The alternate embodiment of the mixing device 100 constructed using thecentral section 5900 depicted in FIG. 13 and the bearing housing 5920depicted in FIG. 14 may be operated in at least two ways. First, theelectrical contacts 5910 and 5922 may be configured not to provide anelectrical charge to the stator 700 and the rotor 600, respectively. Inother words, neither of the electrical contacts 5910 and 5922 areconnected to a current source, a voltage source, and the like.

Alternatively, the electrical contacts 5910 and 5922 may be configuredto provide an electrical charge to the stator 700 and the rotor 600,respectively. For example, the electrical contacts 5910 and 5922 may becoupled to a DC voltage source (not shown) supplying a steady orconstant voltage across the electrical contacts 5910 and 5922. Thenegative terminal of the DC voltage source may be coupled to either ofthe electrical contacts 5910 and 5922 and the positive terminal of theDC voltage source may be coupled to the other of the electrical contacts5910 and 5922. The voltage supplied across the electrical contacts 5910and 5922 may range from about 0.0001 volts to about 1000 volts. Inparticular embodiments, the voltage may range from about 1.8 volts toabout 2.7 volts. By way of another example, a pulsed DC voltage having aduty cycle of between about 1% to about 99% may be used.

While the above examples of methods of operating the mixing device applya DC voltage across the electrical contacts 5910 and 5922, as isapparent to those of ordinary skill in the art, a symmetrical AC voltageor non symmetrical AC voltage having various shapes and magnitudes maybe applied across the electrical contacts 5910 and 5922 and suchembodiments are within the scope of the present invention.

Mixing Inside the Mixing Chamber 330

As mentioned above, in the prior art device 10 (shown in FIG. 1), thefirst material 110 entered the channel 32 between the rotor 12 and thestator 30 via a single limited input port 37 located along only aportion of the open second end of the channel 32. Likewise, the outputmaterial 102 exited the channel 32 via a single limited output port 40located along only a portion of the open first end of the channel 32.This arrangement caused undesirable and unnecessary friction. Byreplacing the single limited inlet port 37 and the single limited outletport 40 with the chambers 310 and 320, respectively, friction has beenreduced. Moreover, the first material 110 does not negotiate a cornerbefore entering the mixing chamber 330 and the output material 102 doesnot negotiate a corner before exiting the mixing chamber 330. Further,the chambers 310 and 320 provide for circumferential velocity of thematerial prior to entering, and after exiting the channel 32.

Accordingly, pressure drop across the mixing device 100 has beensubstantially reduced. In the embodiments depicted in FIGS. 2, 4-9, and11, the pressure drop between the input port 1010 and the output port3010 is only approximately 12 psi when the mixing device 100 isconfigured to produce about 60 gallons of the output material 102 perminute. This is an improvement over the prior art device 10 depicted inFIG. 1, which when producing about 60 gallons of output material perminute was at least 26 psi. In other words, the pressure drop across themixing device 100 is less than half that experienced by the prior artdevice 10.

According to additional aspects, the inclusion of pumps 410 and 420,which are powered by the drive shaft 500, provides a configuration thatis substantially more efficient in mixing materials and that requiresless energy than the external pumps used in the prior art.

Micro-Cavitation

During operation of the mixing device 100, the input materials mayinclude the first material 110 (e.g., a fluid) and the second material120 (e.g., a gas). The first material 110 and the second material 120are mixed inside the mixing chamber 330 formed between the rotor 600 andthe stator 700. Rotation of the rotor 600 inside the stator 700 agitatesthe first material 110 and the second material 120 inside the mixingchamber 330. The through-holes 608 formed in the rotor 600 and/or theapertures 708 formed in the stator 700 impart turbulence in the flow ofthe first material 110 and the second material 120 inside the mixingchamber 330.

Without being limited by theory, the efficiency and persistence of thediffusion of the second material 120 into the first material 110 isbelieved to be caused in part by micro-cavitation, which is described inconnection with FIGS. 15-17. Whenever a material flows over a smoothsurface, a rather laminar flow is established with a thin boundary layerthat is stationary or moving very slowly because of the surface tensionbetween the moving fluid and the stationary surface. The through-holes608 and optionally, the apertures 708, disrupt the laminar flow and cancause localized compression and decompression of the first material 110.If the pressure during the decompression cycle is low enough, voids(cavitation bubbles) will form in the material. The cavitation bubblesgenerate a rotary flow pattern 5990, like a tornado, because thelocalized area of low pressure draws the host material and the infusionmaterial, as shown in FIG. 15. When the cavitation bubbles implode,extremely high pressures result. As two aligned openings (e.g., one ofthe apertures 708 and one of the through-holes 608) pass one another, asuccussion (shock wave) occurs, generating significant energy. Theenergy associated with cavitation and succussion mixes the firstmaterial 110 and the second material 120 together to an extremely highdegree, perhaps at the molecular level.

The tangential velocity of the rotor 600 and the number of openings thatpass each other per rotation may dictate the frequency at which themixing device 100. It has been determined that operating the mixingdevice 100 within in the ultrasonic frequency range can be beneficial inmany applications. It is believed that operating the mixing device 100in the ultrasonic region of frequencies provides the maximum successionshock energy to shift the bonding angle of the fluid molecule, whichenables it to transport an additional quantity of the second material120 which it would not normally be able to retain. When the mixingdevice 100 is used as a diffuser, the frequency at which the mixingdevice 100 operates appears to affect the degree of diffusion, leadingto much longer persistence of the second material 120 (infusionmaterial) in the first material 110 (host material).

Referring now to FIG. 18, an alternate embodiment of the rotor 600,rotor 6000 is provided. The cavitations created within the firstmaterial 110 in the mixing chamber 330 may be configured to occur atdifferent frequencies along the length of the mixing chamber 330. Thefrequencies of the cavitations may be altered by altering the numberand/or the placement of the through-holes 6608 along the length of therotor 600. Each of the through-holes 6608 may be substantially similarto the through-holes 608 (discussed above).

By way of non-limiting example, the rotor 6000 may be subdivided intothree separate exemplary sections 6100, 6200, and 6300. Thethrough-holes 6608 increase in density from the section 6100 to thesection 6200, the number of holes in the section 6100 being greater thanthe number of holes in the section 6200. The through-holes 6608 alsoincrease in density from the section 6200 to the section 6300, thenumber of holes in the section 6200 being greater than the number ofholes in the section 6300. Each of the sections 6100, 6200, and 6300create succussions within their particular area at a different frequencydue to the differing numbers of through-holes 6608 formed therein.

By manufacturing the rotor 6000 with a desired number of through-holes6608 appropriately arranged in a particular area, the desired frequencyof the succussions within the mixing chamber 330 may be determined.Similarly, the desired frequency of the cavitations may be determined bya desired number of apertures 708 appropriately arranged in a particulararea upon the stator 700 within which the rotor 600 rotates. Further,the desired frequency (or frequencies) of the succussions within themixing chamber 330 may be achieved by selecting both a particular numberand arrangement of the apertures 708 formed in the stator 700 and aparticular number and arrangement of the through-holes 608 formed in therotor 600.

FIGS. 19-21, depict various alternative arrangements of the apertures708 formed in the stator 700 and the through-holes 608 formed in therotor 600 configured to achieve different results with respect to thecavitations created. FIG. 19 illustrates a configuration in which theapertures 708 and the through-holes 608 are aligned along an axis 7000that is not parallel with any line (e.g., line 7010) drawn through theaxis of rotation “α” of the rotor 600. In other words, if the rotor 600has a cylindrical shape, the axis 7000 does not pass through the centerof the rotor 600. Thus, the first material 110 within the mixing chamber330 will not be oriented perpendicularly to the compressions anddecompressions created by the apertures 708 and the through-holes 608.The compressions and decompressions will instead have a force vectorthat has at least a component parallel to the circumferential flow (inthe direction of arrow “C3” of FIG. 9) of first material 110 within themixing chamber 330.

Relative alignment of the apertures 708 and the through-holes 608 mayalso affect the creation of cavitations in the mixing chamber 330. FIG.20 illustrates an embodiment in which the apertures 708 are inregistration across the mixing chamber 330 with the through-holes 608.In this embodiment, rotation of the rotor 600 brings the through-holes608 of the rotor into direct alignment with the apertures 708 of thestator 700. When in direct alignment with each other, the compressiveand decompressive forces created by the apertures 708 and thethrough-holes 608 are directly aligned with one another.

In the embodiment depicted in FIG. 21, the apertures 708 and thethrough-holes 608 are offset by an offset amount “X” along the axis ofrotation “α.”. By way of non-limiting example, the offset amount “X” maybe determined as a function of the size of the apertures 708. Forexample, the offset amount “X” may be approximately equal to one half ofthe diameter of the apertures 708. Alternatively, the offset amount “X”may be determined as a function of the size of the through-holes 608.For example, the offset amount “X” may be approximately equal to onehalf of the diameter of the through-holes 608. If features (e.g.,recesses, projections, etc.) other than or in addition to thethrough-holes 608 and the apertures 708 are included in either the rotor600 or the stator 700, the offset amount “X” may be determined as afunction of the size of such features. In this manner, the compressiveand decompressive forces caused by the apertures 708 of the stator 700and the through-holes 608 of the rotor 600 collide at a slight offsetcausing additional rotational and torsional forces within the mixingchamber 330. These additional forces increase the mixing (e.g.,diffusive action) of the second material 120 into the first material 110within the mixing chamber 330.

Referring now to FIGS. 22-25, non-limiting examples of suitablecross-sectional shapes for the apertures 708 and the through-holes 608are provided. The cross-sectional shape of the apertures 708 and/or thethrough-holes 608 may be square as illustrated in FIG. 22, circular asillustrated in FIG. 23, and the like.

Various cross-sectional shapes of apertures 708 and/or the through-holes608 may be used to alter flow of the first material 110 as the rotor 600rotates within the stator 700. For example, FIG. 24 depicts a teardropcross-sectional shape having a narrow portion 7020 opposite a wideportion 7022. If the through-holes 608 have this teardrop shape, whenthe rotor 600 is rotated (in the direction generally indicated by thearrow “F”), the forces exerted on the first material 110, the secondmaterial 120, and optionally the third material 130 within the mixingchamber 330 increase as the materials pass from the wide portion 7022 ofthe teardrop to the narrow portion 7020.

Additional rotational forces can be introduced into the mixing chamber330 by forming the apertures 708 and/or the through-holes 608 with aspiral configuration as illustrated in FIG. 25. Material that flows intoand out of the apertures 708 and/or the through-holes 608 having thespiral configuration experience a rotational force induced by the spiralconfiguration. The examples illustrated in FIGS. 22-25 are provided asnon-limiting illustrations of alternate embodiments that may be employedwithin the mixing device 100. By application of ordinary skill in theart, the apertures 708 and/or the through-holes 608 may be configured innumerous ways to achieve various succussive and agitative forcesappropriate for mixing materials within the mixing chamber 330.

Double Layer Effect

The mixing device 100 may be configured to create the output material102 by complex and non-linear fluid dynamic interaction of the firstmaterial 110 and the second material 120 with complex, dynamicturbulence providing complex mixing that further favors electrokineticeffects (described below). The result of these electrokinetic effectsmay be observed within the output material 102 as charge redistributionsand redox reactions, including in the form of solvated electrons thatare stabilized within the output material.

Ionization or dissociation of surface groups and/or adsorption of ionsfrom a liquid cause most solid surfaces in contact with the liquid tobecome charged. Referring to FIG. 26, an electrical double layer (“EDL”)7100 forms around exemplary surface 7110 in contact with a liquid 7120.In the EDL 7100, ions 7122 of one charge (in this case, negativelycharged ions) adsorb to the surface 7120 and form a surface layer 7124typically referred to as a Stern layer. The surface layer 7124 attractscounterions 7126 (in this case, positively charged ions) of the oppositecharge and equal magnitude, which form a counterion layer 7128 below thesurface layer 7124 typically referred to as a diffuse layer. Thecounterion layer 7128 is more diffusely distributed than the surfacelayer 7124 and sits upon a uniform and equal distribution of both ionsin the bulk material 7130 below. For OH− and H+ ions in neutral water,the Gouy-Chapman model would suggest that the diffuse counterion layerextends about one micron into the water.

According to particular aspects, the electrokinetic effects mentionedabove are caused by the movement of the liquid 7120 next to the chargedsurface 7110. Within the liquid 7120 (e.g., water, saline solution, andthe like), the adsorbed ions 7122 forming the surface layer 7124 arefixed to the surface 7120 even when the liquid 7120 is in motion (forexample, flowing in the direction indicated by arrow “G”); however, ashearing plane 7132 exists within the diffuse counterion layer 7128spaced from the surface 7120. Thus, as the liquid 7120 moves, some ofthe diffuse counterions 7126 are transported away from the surface 7120,while the absorbed ions 7122 remain at the surface 7120. This produces aso-called ‘streaming current.’

Within the mixing chamber 330, the first material 110, the secondmaterial 120, and optionally, the third material 130 are subject to anelectromagnetic field created by the inside surface 705 of the stator700 and/or the outside surface 606 of the rotor 600, a voltage betweenthe inside surface 705 and the outside surface 606, and/or anelectrokinetic effect (e.g., streaming current) caused by at least oneEDL formed in the first material 110. The at least one EDL may beintroduced into the first material 110 by at least one of the insidesurface 705 of the stator 700 and the outside surface 606 of the rotor600.

Movement of the first material 110 through the mixing chamber 330relative to surface disturbances (e.g., the through-holes 608 andapertures 708) creates cavitations in the first material 110 within themixing chamber 330, which may diffuse the second material 120 into thefirst material 110. These cavitations may enhance contact between of thefirst material 110 and/or the second material 120 with the electricdouble layer formed on the inside surface 705 of the stator 700 and/orthe electric double layer formed on the outside surface 606 of the rotor600. Larger surface to volume ratios of the mixing chamber, an increaseddwell time of the combined materials within the mixing chamber, andfurther in combination with a smaller average bubble size (and hencesubstantially greater bubble surface area) provide for effectivelyimparting EDL-mediated effects to the inventive output materials.

In embodiments in which the inside surface 705 and the outside surface606 are constructed from a metallic material, such as stainless steel,the motion of the liquid 7120 and/or the streaming current(s) facilitateredox reactions involving H₂O, OH−, H+, and O₂ at the inside surface 705and the outside surface 606.

Referring to FIG. 27, without being limited by theory, it is believed asection 7140 of the mixing chamber 330 between the inside surface 705and the outside surface 606 the may be modeled as a pair of parallelplates 7142 and 7144. If the first material 110 is a liquid, the firstmaterial 110 enters the section 7140 through an inlet “IN” and exits thesection 7140 through an outlet “OUT.” The inlet “IN” and the outlet“OUT” restrict the flow into and out of the section 7140.

Referring to FIG. 28, the area between the parallel plates 7142 and 7144has a high surface area to volume ratio. Hence, a substantial portion ofthe counterion layer 7128 (and counterions 7126) may be in motion as thefirst material 110 moves between the plates 7142 and 7144. The number ofcounterions 7126 in motion may exceed the number allowed to enter thesection 7140 by the inlet “IN” and the number allowed to exit thesection 7140 by the outlet “OUT.” The inlet “IN” and the outlet “OUT”feeding and removing the first material 110 from the section 7140,respectively, have far less surface area (and a lower surface area tovolume ratio) than the parallel plates 7142 and 7144 and thereby reducethe portion of the counterions 7126 in motion in the first material 110entering and leaving the section 7140. Therefore, entry and exit fromthe section 7140 increases the streaming current locally. While abackground streaming current (identified by arrow “BSC”) caused by theflowing first material 110 over any surface is always present inside themixing device 100, the plates 7142 and 7144 introduce an increased“excess” streaming current (identified by arrow “ESC”) within thesection 7140.

Without a conductive return current (identified by arrow “RC”) in theplates 7142 and 7144 in the opposite direction of the flow of the firstmaterial 110, an excess charge 7146 having the same sign as theadsorbing ions 7122 would accumulate near the inlet “IN,” and an excesscharge 7148 having the same sign as the counterion 7126 would accumulatenear the at outlet “OUT.” Because such accumulated charges 7146 and7148, being opposite and therefore attracted to one another, cannotbuild up indefinitely the accumulated charges seek to join together byconductive means. If the plates 7142 and 7144 are perfectly electricallyinsulating, the accumulated charges 7146 and 7148 can relocate onlythrough the first material 110 itself. When the conductive returncurrent (identified by arrow “RC”) is substantially equivalent to theexcess streaming current (identified by arrow “ESC”) in the section7140, a steady-state is achieved having zero net excess streamingcurrent, and an electrostatic potential difference between the excesscharge 7146 near the inlet “IN,” and the excess charge 7148 near theoutlet “OUT” creating a steady-state charge separation therebetween.

The amount of charge separation, and hence the electrostatic potentialdifference between the excess charge 7146 near the inlet “IN,” and theexcess charge 7148 near the outlet “OUT,” depends on additional energyper unit charge supplied by a pump (e.g., the rotor 600, the internalpump 410, and/or the external pump 210) to “push” charge against theopposing electric field (created by the charge separation) to producethe a liquid flow rate approximating a flow rate obtainable by a liquidwithout ions (i.e., ions 7122 and 7126). If the plates 7142 and 7144 areinsulators, the electrostatic potential difference is a direct measureof the EMF the pump (e.g., the rotor 600, the internal pump 410 and/orthe external pump 210) can generate. In this case, one could measure theelectrostatic potential difference using a voltmeter having a pair ofleads by placing one of the leads in the first material 110 near theinlet “IN,” and the other lead in the first material 110 near the outlet“OUT.”

With insulating plates 7142 and 7144, any return current is purely anion current (or flow of ions), in that the return current involves onlythe conduction of ions through the first material 110. If otherconductive mechanisms through more conductive pathways are presentbetween the excess charge 7146 near the inlet “IN,” and the excesscharge 7148 near the outlet “OUT,” the return current may use those moreconductive pathways. For example, conducting metal plates 7142 and 7144may provide more conductive pathways; however, these more conductivepathways transmit only an electron current and not the ion current.

As is appreciated by those of ordinary skill, to transfer the chargecarried by an ion to one or more electrons in the metal, and vise versa,one or more oxidation-reduction reactions must occur at the surface ofthe metal, producing reaction products. Assuming the first material 110is water (H₂O) and the second material 120 is oxygen (O₂), anon-limiting example of a redox reaction, which would inject negativecharge into the conducting plates 7142 and 7144 includes the followingknown half-cell reaction:

O₂+H₂O→O₃+2H⁺+2e ⁻,

Again, assuming the first material 110 is water (H₂O) and the secondmaterial 120 is oxygen (O₂), a non-limiting example of a redox reactionincludes the following known half-cell reaction, which would removenegative charge from the conducting plates 7142 and 7144 includes thefollowing known half-cell reaction:

2H⁺ +e ⁻→H₂,

With conducting metal plates 7142 and 7144, most of the return currentis believed to be an electron current, because the conducting plates7142 and 7144 are more conductive than the first material 110 (providedthe redox reactions are fast enough not to be a limiting factor). Forthe conducting metal plates 7142 and 7144, a smaller charge separationaccumulates between the inlet “IN” and the outlet “OUT,” and a muchsmaller electrostatic potential exists therebetween. However, this doesnot mean that the EMF is smaller.

As described above, the EMF is related to the energy per unit charge thepump provides to facilitate the flow of the first material 110 againstthe opposing electric field created by the charge separation. Becausethe electrostatic potential is smaller, the pump may supply less energyper unit charge to cause the first material 110 to flow. However, theabove example redox reactions do not necessarily occur spontaneously,and thus may require a work input, which may be provided by the pump.Therefore, a portion of the EMF (that is not reflected in the smallerelectrostatic potential difference) may be used to provide the energynecessary to drive the redox reactions.

In other words, the same pressure differentials provided by the pump topush against the opposing electric field created by the chargeseparation for the insulating plates 7142 and 7144, may be used both to“push” the charge through the conducting plates 7142 and 7144 and drivethe redox reactions.

Referring to FIG. 29, an experimental setup for an experiment conductedby the inventors is provided. The experiment included a pair ofsubstantially identical spaced apart 500 ml standard Erlenmeyer flasks7150 and 7152, each containing a volume of deionized water 7153. Arubber stopper 7154 was inserted in the open end of each of the flasks7150 and 7152. The stopper 7154 included three pathways, one each for ahollow tube 7156, a positive electrode 7158, and a negative electrode7160. With respect to each of the flasks 7150 and 7152, each of thehollow tube 7156, the positive electrode 7158, and the negativeelectrode 7160 all extended from outside the flask, through the stopper7154, and into the deionized water 7153 inside the flask. The positiveelectrode 7158 and the negative electrode 7160 were constructed fromstainless steel. The hollow tubes 7156 in both of the flasks 7150 and7152 had an open end portion 7162 coupled to a common oxygen supply7164. The positive electrode 7158 and the negative electrode 7160inserted into the flask 7152 where coupled to a positive terminal and anegative terminal, respectively, of a DC power supply 7168. Exactly thesame sparger was used in each flask.

Oxygen flowed through the hollow tubes 7156 into both of the flasks 7150and 7152 at a flow rate (Feed) of about 1 SCFH to about 1.3 SCFH(combined flow rate). The voltage applied across the positive electrode7158 and the negative electrode 7160 inserted into the flask 7152 wasabout 2.55 volts. This value was chosen because it is believed to be anelectrochemical voltage value sufficient to affect all oxygen species.This voltage was applied continuously over three to four hours duringwhich oxygen from the supply 7164 was bubbled into the deionized water7153 in each of the flasks 7150 and 7152.

Testing of the deionized water 7153 in the flask 7150 with HRP andpyrogallol gave an HRP-mediated pyrogallol reaction activity, consistentwith the properties of fluids produced with the alternate rotor/statorembodiments described herein. The HRP optical density was about 20%higher relative to pressure-pot or fine-bubbled solutions of equivalentoxygen content. The results of this experiment indicate that mixinginside the mixing chamber 330 involves a redox reaction. According toparticular aspects, the inventive mixing chambers provide for outputmaterials comprising added electrons that are stabilized by eitheroxygen-rich water structure within the inventive output solutions, or bysome form of oxygen species present due to the electrical effects withinthe process.

Additionally, the deionized water 7153 in both of the flasks 7150 and7152 was tested for both ozone and hydrogen peroxide employing industrystandard colorimetric test ampoules with a sensitivity of 0.1 ppm forhydrogen peroxide and 0.6 ppm for ozone. There was no positiveindication of either species up to the detection limits of thoseampoules.

Dwell Time

Dwell time is an amount of time the first material 110, the secondmaterial 120, and optionally the third material 130 spend in the mixingchamber 330. The ratio of the length of the mixing chamber 330 to thediameter of the mixing chamber 330 may significantly affect dwell time.The greater the ratio, the longer the dwell time. As mentioned in theBackground Section, the rotor 12 of the prior art device 10 (see FIG. 1)had a diameter of about 7.500 inches and a length of about 6.000 inchesproviding a length to diameter ratio of about 0.8. In contrast, inparticular embodiments, the length of the mixing chamber 330 of themixing device 100 is about 5 inches and the diameter “D1” of the rotor600 is about 1.69 inches yielding a length to diameter ratio of about2.95.

Dwell time represents the amount of time that the first material 110,the second material 120, and optionally the third material 130 are ableto interact with the electrokinetic phenomena described herein. Theprior art device 10 is configured to produce about 60 gallons of theoutput material 102 per minute and the mixing device 100 is configuredto produce about 0.5 gallons of the output material 102 per minute, theprior art device 10 (see FIG. 1) had a fluid dwell time of about 0.05seconds, whereas embodiments of the mixing device 100 have asubstantially greater (about 7-times greater) dwell time of about 0.35seconds. This longer dwell time allows the first material 110, thesecond material 120, and optionally the third material 130 to interactwith each other and the surfaces 606 and 705 (see FIG. 7) inside themixing chamber 330 for about 7 times longer than was possible in theprior art device 10.

With reference to Table I below, the above dwell times were calculatedby first determining the flow rate for each device in gallons persecond. In the case of the prior art device 10 was configured to operateat about 60 gallons of output material per minute, while the mixingdevice 100 is configured to operate over a broader range of flow rate,including at an optimal range of about 0.5 gallons of output materialper minute. The flow rate was then converted to cubic inches per secondby multiplying the flow rate in gallons per second by the number ofcubic inches in a gallon (i.e., 231 cubic inches). Then, the volume(12.876 cubic inches) of the channel 32 of the prior art device 10 wasdivided by the flow rate of the device (231 cubic inches/second) toobtain the dwell time (in seconds) and the volume (0.673 cubic inches)of the mixing chamber 330 of the mixing device 100 was divided by theflow rate (1.925 cubic inches/second) of the device (in cubic inches persecond) to obtain the dwell time (in seconds).

TABLE 1 Inventive device can accommodate a range of dwell times,including a substantially increased (e.g., 7-times) dwell time relativeto prior art devices. Volume Flow Rate Mixing Flow Rate Flow Rate CubicChamber Dwell Gallons/ Gallons/ Inches/ (Cubic Time Device Minute SecondSecond Inches) (Seconds) Prior art 60 1.000 231.000 12.876 0.056 device10 Mixing 2 0.033 7.700 0.673 0.087 device 100 Mixing 0.5 0.008 1.9250.673 0.350 device 100

Rate of Infusion

Particular aspects of the mixing device 100 provide an improved oxygeninfusion rate over the prior art, including over prior art device 10(see FIG. 1). When the first material 110 is water and the secondmaterial 120 is oxygen, both of which are processed by the mixing device100 in a single pass (i.e., the return block of FIG. 2 is set to “NO”)at or near 20° Celsius, the output material 102 has a dissolved oxygenlevel of about 43.8 parts per million. In certain aspects, an outputmaterial having about 43.8 ppm dissolved oxygen is created in about 350milliseconds via the inventive flow through the inventive nonpressurized (non-pressure pot) methods. In contrast, when the firstmaterial 110 (water) and the second material 120 (oxygen) are bothprocessed in a single pass at or near 20° Celsius by the prior artdevice 10, the output material had dissolved oxygen level of only 35parts per million in a single pass of 56 milliseconds.

Output Material 102

When the first material 110 is a liquid (e.g., freshwater, saline,GATORADE®, and the like) and the second material 120 is a gas (e.g.,oxygen, nitrogen, and the like), the mixing device 100 may diffuse thesecond material 120 into the first material 110. The following discussesresults of analyses performed on the output material 102 to characterizeone or more properties of the output material 102 derived from havingbeen processed by the mixing device 100.

When the first material 110 is saline solution and the second material120 is oxygen gas, experiments have indicated that a vast majority ofoxygen bubbles produced within the saline solution are no greater than0.1 micron in size.

Decay of Dissolved Oxygen Levels

Referring now to FIG. 30, there is illustrated the DO levels in waterenriched with oxygen in the mixing device 100 and stored in a 500 mlthin-walled plastic bottle and a 1000 ml glass bottle out to at least365 days. Each of the bottles was capped and stored at 65 degreesFahrenheit. As can be seen in the Figure, the DO levels of theoxygen-enriched fluid remained fairly constant out to at least 365 days.

Referring to FIG. 31, there is illustrated the DO levels in waterenriched with oxygen in the mixing device 100 and stored in a 500 mlplastic thin-walled bottle and a 1000 ml glass bottle. Both bottles wererefrigerated at 39 degrees Fahrenheit. Again, DO levels of theoxygen-enriched fluid remained steady and decreased only slightly out toat least 365 days.

Referring now to FIG. 32, there is illustrated the dissolved oxygenlevels in GATORADE® enriched with oxygen in the mixing device 100 andstored in 32 oz. GATORADE® bottles having an average temperature of 55degrees Fahrenheit at capping. The GATORADE® bottles were subsequentlyrefrigerated at 38 degrees Fahrenheit between capping and opening.During the experiment, a different bottle was opened at 20, 60, and 90days, respectively, to measure the DO levels of the GATORADE® storedtherein.

The GATORADE® within a first group of GATORADE® bottles was processedwith oxygen in the mixing device 100 at approximately 56 degreesFahrenheit. The DO levels of the GATORADE® at bottling wereapproximately 50 ppm as indicated by point 8104. A first bottle wasopened at approximately 20 days, and the DO level of the GATORADE® wasdetermined to be approximately 47 ppm as indicated by point 8106. Asecond bottle was then opened at 60 days, and the DO level of theGATORADE® was measured to be approximately 44 ppm as indicated by point8108. Finally, a third bottle was opened at 90 days, and the DO level ofthe GATORADE® was determined to be slightly below 40 ppm as indicated bypoint 8110.

The GATORADE® within a second group of GATORADE® bottles was processedwith oxygen in the mixing device 100 at approximately 52 degreesFahrenheit. The initial DO level for GATORADE® stored in this group ofbottles was 45 ppm as illustrated by point 8112. The GATORADE® in thebottle opened at 20 days had a DO level of only slightly lower than 45ppm as indicated by point 8114. The second bottle of GATORADE® wasopened at 60 days and the GATORADE® therein had a DO level of slightlymore than 41 ppm. Finally, a third bottle of GATORADE® was opened at 90days and the GATORADE® therein had a DO level of approximately 39 ppm asshown by point 8116. As before, with respect to the water test in theplastic and glass bottles (see FIG. 31), it can be seen that the DOlevels remain at relatively high levels over the 90 day period andsubstantially higher than those levels present in normal (unprocessed)GATORADE® stored in 32 oz. GATORADE® bottles. Point 8010 is the levelcorresponding to inventive output fluid in a covered PET bottle.

FIG. 33 illustrates the DO retention of 500 ml of braun balanced saltsolution processed with oxygen in the mixing device 100 and kept atstandard temperature and pressure in an amber glass bottle. The DO levelof the solution before processing is 5 ppm. After processing in themixing device 100, the DO level was increased to approximately 41 ppm(illustrated as point 8202). An hour after processing, the DO leveldropped to approximately 40 ppm as indicated by point 8204. Two hoursafter processing, the DO level dropped to approximately 36 ppm asindicated by point 8206. The DO level dropped to approximately 34 ppmthree hours after processing as indicated by point 8208. Atapproximately four and a half hours after processing, the DO levelwithin the salt solution dropped to slightly more than 30 ppm. The finalmeasurement was taken shortly before six hours after processing whereatthe DO level had dropped to approximately 28 ppm. Thus, each of theexperiments illustrated in FIGS. 30-33 illustrate that that the DOlevels remain at relatively high levels over extended periods.

Because the output material 102 may be consumed by human beings, thematerials used to construct the mixing device 100 should be suitable forfood and/or pharmaceutical manufacture. By way of non-limiting example,the housing 520, the housing 5520, the rotor 600, the stator 700, andthe stator 5700 may all be constructed from stainless steel.

Inventive Electrokinetically-Generated Gas-Enriched Fluids and Solutions

Diffusing or enriching a fluid with another fluid may result in asolution or suspension of the two fluids. In particular, enriching aliquid with a gas (e.g. oxygen) may be beneficial for certainapplications, including therapeutic treatments. As utilized herein,“fluid,” may generally refer to a liquid, a gas, a vapor, a mixture ofliquids and/or gases, or any combination thereof, for any particulardisclosed embodiment. Furthermore, in certain embodiments a “liquid” maygenerally refer to a pure liquid or may refer to a gel, sol, emulsion,fluid, colloid, dispersion, or mixture, as well as any combinationthereof; any of which may vary in viscosity.

In particular embodiments disclosed herein, the dissolved gas comprisesambient air. In a preferred embodiment, the dissolved gas comprisesoxygen. In another embodiment, the dissolved gas comprises nitric oxide.

There are several art-recognized methods of gas-enriching liquids (suchas oxygen-enriching water). For example, a turbine aeration system canrelease air near a set of rotating blades of an impeller, which mixesthe air or oxygen with the water, or water can be sprayed into the airto increase its oxygen content. Additionally, other systems on themarket inject air or oxygen into the water and subject the water/gas toa large-scale vortex. Naturally occurring levels of oxygen in water aretypically no more than 10 ppm (parts per million), which is consideredto be a level of 100% dissolved oxygen. Tests on certain devices haveshown that under ideal conditions, the device can attain upwards ofapproximately 20 ppm, or twice the natural oxygen levels of water. Incertain embodiments, the oxygen level may be even higher.

In certain embodiments disclosed herein, a gas-enriched fluid of thepresent invention provides an anti-inflammatory benefit. Certainembodiments disclosed herein relate to a therapeutic compositioncomprising a gas-enriched fluid of the present invention, and optionallyat least one additional therapeutic agent, such as a pharmaceuticaldrug, a metal, a peptide, a polypeptide, a protein, a nucleotide, acarbohydrate or glycosylated protein, a fat (including oils or waxes),or other agent that prevents or alleviates at least one symptom of acondition or disease associated with inflammation.

Furthermore, certain embodiments disclosed herein include therapeuticcompositions and methods related to inflammation of wounds. Wound careis desirable to improve health and appearance of underlying dermaltissues. Wounds, either injury induced, such as cuts, abrasions orblisters, or surgically induced, such as surgical incisions or ostomies,require localized treatment to remedy the affected area and to preventfurther dermal damage. If wounds are not properly treated, furtherdermal irritation can result, such as inflammation, and may result insecondary infections and further discomfort to the subject.

Particular embodiments provided herein relate to a diffuser-processedtherapeutic fluid as defined herein, comprising: a fluid host material;an infusion material diffused into the host material; and optionally, atleast one therapeutic agent dispersed in the host material, wherein theinfusion material comprises oxygen micro-bubbles in the host fluid,wherein the majority of the micro-bubbles are less than 0.2 microns, orpreferably less than 0.1 microns in size. In certain embodiments, thedissolved oxygen level in the infused fluid host material may bemaintained at greater than about 30 ppm at atmospheric pressure for atleast 13 hours. In other particular embodiments, the dissolved oxygenlevel in the infused fluid host material may be maintained at greaterthan 40 ppm at atmospheric pressure for at least 3 hours.

In additional embodiments, the infused fluid host material furthercomprises a saline solution. In further embodiments, the infused fluidhost material maintains a dissolved oxygen level of at least about 20ppm to about 40 ppm for a period of at least 100 days, preferably atleast 365 days within a sealed container at atmospheric pressure. Incertain embodiments, the infused fluid host material may have adissolved oxygen level of at least 50 ppm at atmospheric pressure.

In certain embodiments, the infused fluid host material exhibitsRayleigh scattering for a laser beam shining therethrough for a selectedperiod of time after the oxygen has been diffused into therein.

Table 2 illustrates various partial pressure measurements taken in ahealing wound treated with an oxygen-enriched saline solution and insamples of the gas-enriched oxygen-enriched saline solution of thepresent invention.

TABLE 2 TISSUE OXYGEN MEASUREMENTS Probe Z082BO TISSUE OXYGENMEASUREMENTS In air: 171 mmHg 23° C. Column Partial Pressure (mmHg) B132-36 B2 169-200 B3  20-180* B4 40-60 *wound depth minimal,majority >150, occasional 20 s

Bubble Size Measurements

Experimentation was performed to determine a size of the bubbles of gasdiffused within the fluid by the mixing device 100. While experimentswere not performed to measure directly the size of the bubbles,experiments were performed that established that the bubble size of themajority of the gas bubbles within the fluid was smaller than 0.1microns. In other words, the experiments determined a size thresholdvalue below which the sizes of the majority of bubbles fall.

This size threshold value or size limit was established by passing theoutput material 102 formed by processing a fluid and a gas in the mixingdevice 100 through a 0.22 filter and a 0.1 micron filter. In performingthese tests, a volume of the first material 110, in this case, a fluid,and a volume of the second material 120, in this case, a gas, werepassed through the mixing device 100 to generate a volume of the outputmaterial 102 (i.e., a fluid having a gas diffused therein). Sixtymilliliters of the output material 102 was drained into a 60 ml syringe.The DO level of the fluid was measured via the Winkler Titration. Thefluid within the syringe was injected through a 0.22 micron filter intoa 50 ml beaker. The filter comprised the Milipor Millex GP50 filter. TheDO level of the material in the 50 ml beaker was then measured. Theexperiment was performed three times to achieve the results illustratedin Table 3 below.

TABLE 3 DO levels DO AFTER 0.22 MICRON DO IN SYRINGE FILTER 42.1 ppm39.7 ppm 43.4 ppm 42.0 ppm 43.5 ppm 39.5 ppm

As can be seen, the DO levels measured within the syringe and the DOlevels measured within the 50 ml beaker were not changed drastically bypassing the output material 102 through the 0.22 micron filter. Theimplication of this experiment is that the bubbles of dissolved gaswithin the output material 102 are not larger than 0.22 micronsotherwise there would be a significantly greater reduction in the DOlevels in the output material 102 passed through the 0.22 micron filter.

A second test was performed in which the 0.1 micron filter wassubstituted for the 0.22 micron filter. In this experiment, salinesolution was processed with oxygen in the mixing device 100 and a sampleof the output material 102 was collected in an unfiltered state. The DOlevel of the unfiltered sample was 44.7 ppm. The output material 102 wasfiltered using the 0.1 micron filter and two additional samples werecollected. The DO level of the first sample was 43.4 ppm. The DO levelof the second sample was 41.4 ppm. Then, the filter was removed and afinal sample was taken from the unfiltered output material 102. Thefinal sample had a DO level of 45.4 ppm. These results were consistentwith those seen using the Millipore 0.22 micron filter. These resultslead to the conclusion that there is a trivial reduction in the DOlevels of the output material 102 passed through the 0.1 micron filterproviding an indication that the majority of the bubbles in theprocessed saline solution are no greater than 0.1 micron in size.

As appreciated in the art, the double-layer (interfacial) (DL) appearson the surface of an object when it is placed into a liquid. Thisobject, for example, might be that of a solid surface (e.g., rotor andstator surfaces), solid particles, gas bubbles, liquid droplets, orporous body. In the mixing device 100, bubble surfaces represent asignificant portion of the total surface area present within the mixingchamber that may be available for electrokinetic double-layer effects.Therefore, in addition to the surface area and retention time aspectsdiscussed elsewhere herein, the relatively small bubble sizes generatedwithin the mixer 100 compared to prior art devices 10, may alsocontribute, at least to some extent, to the overall electrokineticeffects and output fluid properties disclosed herein. Specifically, inpreferred embodiments, as illustrated by the mixer 100, all of the gasis being introduced via apertures on the rotor (no gas is beingintroduced through stator apertures. Because the rotor is rotating at ahigh rate (e.g., 3,400 rpm) generating substantial shear forces at andnear the rotor surface, the bubble size of bubbles introduced via, andadjacent to the spinning rotor surface apertures would be expected to besubstantially (e.g., 2 to 3-times smaller) smaller than those introducedvia and near the stationary stator. The average bubble size of the priorart device 10 may, therefore, be substantially larger because at leasthalf of the gas is introduced into the mixing chamber from thestationary stator apertures. Because the surface area of a spheresurface varies with r², any such bubble component of the electrokineticsurface area of the mixing device 100 may be substantially greater thanthat of the prior art diffusion device 10.

Therefore, without being bound by theory, not only does the mixingchamber of the mixing device 100 have (i) a substantially higher surfaceto volume ratio than that of the prior art device 10 (the prior artdevice 10 has a ratio of surface to volume of 10.9, whereas the presentmixer 100 has a surface to volume ratio of 39.4), along with (ii) a7-fold greater dwell-time, but (iii) the unique properties of thecurrent output solutions may additionally reflect a contribution fromthe substantially larger bubble surface area in the mixing device 100.These distinguishing aspects reflect distinguishing features of thepresent mixing device 100, and likely each contribute to the uniqueelectrokinetic properties of the inventive output materials/fluids.

Sparging Effects

FIGS. 34-35 illustrate the sparging affects of the mixing device 100 ona fluid (e.g., the first material 110) passing therethrough. Spargingrefers to “bubbling” an inert gas through a solution to remove adifferent dissolved gas(es) from the solution. In each of the examplesillustrated in FIGS. 34 and 35, the second material 120 is nitrogen. Thelevels of dissolved oxygen in the output material 102 are measured atvarious points in time. As can be seen in the figures, the nitrogen gassparges the oxygen from the fluid passing through the mixing device 100causing the DO levels in the fluid to decay over a period of time.

The results of another experiment are illustrated in FIG. 34 whereinwater is sparged with nitrogen using the mixing device 100. Two sets ofexperiments were conducted, the first having a gas flow rate of SCFH(Standard Cubic Feet per Hour) of 1 and the second having a gas flowrate of SCFH of 0.6 The fluid flow rate was about 0.5 gal/min. As can beseen, when the process is begun, the DO levels in each of theexperiments was approximately 9 ppm. After only one minute, the DOlevels had dropped to slightly above 5 ppm. At two minutes the DO levelshad dropped to approximately 2.5 ppm. The DO level appears to level outat a minimum level at approximately 6 minutes wherein the DO level isslightly above zero (0). Thus, the nitrogen sparges the oxygen from thewater relatively quickly.

FIG. 35 illustrates the sparging of oxygenated water in an 8 gallon tankat standard temperature and pressure. The decay rate of the DO in thewater is illustrated by line 8602. As can be seen, initially theoxygenated water had a DO level of approximately 42 ppm. After 2 minutesof processing by the mixing device 100, the nitrogen sparged theoxygenated water such that the DO level dropped to slightly more than 20ppm. At 6 minutes, the DO level dropped from greater than 40 ppm to only6 ppm. The DO level of the oxygenated water reached a minimum valueslightly greater than zero (0) at approximately 14 minutes after thebeginning of the process. Thus, the above described sparging experimentsillustrate that the mixing device 100 is capable of quickly spargingoxygen from water and replacing the oxygen with another gas such asnitrogen by processing oxygenated water with mixing device 100 for arather short period of time. In other words, because total partial gaspressure in the fluid remained at approximately the same level despitethe decrease in DO, the nitrogen gas replaced the oxygen in the fluid.

These figures illustrate the manner in which nitrogen may be diffusedinto water to sparge the oxygen from the water. However, any gas couldbe used to sparge a selected gas from any selected fluid and diffuseinto the selected fluid the gas used to sparge the selected gas from theselected fluid. For example, the principals illustrated may also beapplicable to sparging nitrogen from water or another fluid usingoxygen. Further, any gas dissolved within a solution may be spargedtherefrom using a different gas to take the place of the gas spargedfrom the solution. In other words, by processing a sparging gas and asolution containing a dissolved gas through the mixing device 100 for arelatively short period of time, the dissolved gas could be quickly andefficiently removed from the solution.

Molecular Interactions

Conventionally, quantum properties are thought to belong to elementaryparticles of less than 10⁻¹⁰ meters, while the macroscopic world of oureveryday life is referred to as classical, in that it behaves accordingto Newton's laws of motion.

Recently, molecules have been described as forming clusters thatincrease in size with dilution. These clusters measure severalmicrometers in diameter, and have been reported to increase in sizenon-linearly with dilution. Quantum coherent domains measuring 100nanometers in diameter have been postulated to arise in pure water, andcollective vibrations of water molecules in the coherent domain mayeventually become phase locked to electromagnetic field fluctuations,providing for stable oscillations in water, providing a form of ‘memory’in the form of excitation of long lasting coherent oscillations specificto dissolved substances in the watet that change the collectivestructure of the water, which may in turn determine the specificcoherent oscillations that develop. Where these oscillations becomestabilized by magnetic field phase coupling, the water, upon diluctionmay still carry ‘seed’ coherent oscillations. As a cluster of moleculesincreases in size, its electromagnetic signature is correspondinglyamplified, reinforcing the coherent oscillations carried by the water.

Despite variations in the cluster size of dissolved molecules anddetailed microscopic structure of the water, a specificity of coherentoscillations may nonetheless exist. One model for considering changes inproperties of water is based on considerations involved incrystallization.

With reference to FIG. 36, a simplified protonated water cluster forminga nanoscale cage 8700 is shown. A protonated water cluster typicallytakes the form of H⁺(H₂O)_(n). Some protonated water clusters occurnaturally, such as in the ionosphere. Without being bound by anyparticular theory, and according to particular aspects, other types ofwater clusters or structures (clusters, nanocages, etc) are possible,including structures comprising oxygen and stabilized electrons impartedto the inventive output materials. Oxygen atoms 8704 may be caught inthe resulting structures 8700. The chemistry of the semi-bound nanocageallows the oxygen 8704 and/or stabilized electrons to remain dissolvedfor extended periods of time. Other atoms or molecules, such asmedicinal compounds, can be caged for sustained delivery purposes. Thespecific chemistry of the solution material and dissolved compoundsdepend on the interactions of those materials.

Fluids processed by the mixing device 100 have been shown viaexperiments to exhibit different structural characteristics that areconsistent with an analysis of the fluid in the context of a clusterstructure.

Water processed through the mixing device 100 has been demonstrated tohave detectible structural differences when compared with normalunprocessed water. For example, processed water has been shown to havemore Rayleigh scattering than is observed in unprocessed water. In theexperiments that were conducted, samples of processed and unprocessedwater were prepared (by sealing each in a separate bottle), coded (forlater identification of the processed sample and unprocessed sample),and sent to an independent testing laboratory for analysis. Only afterthe tests were completed were the codes interpreted to reveal whichsample had been processed by the mixing device 100.

At the laboratory, the two samples were placed in a laser beam having awavelength of 633 nanometers. The fluid had been sealed in glass bottlesfor approximately one week before testing. With respect to the processedsample, Sample B scattered light regardless of its position relative tothe laser source. However, Sample A did not. After two to three hoursfollowing the opening of the bottle, the scattering effect of Sample Bdisappeared. These results imply the water exhibited a memory causingthe water to retain its properties and dissipate over time. Theseresults also imply the structure of the processed water is opticallydifferent from the structure of the unprocessed fluid. Finally, theseresults imply the optical effect is not directly related to DO levelsbecause the DO level at the start was 45 ppm and at the end of theexperiment was estimated to be approximately 32 ppm.

Charge-Stabilized Nanostructures (e.g. Charge StabilizedOxygen-Containing Nanostructures):

As described herein above under “Double Layer Effect,” “Dwell Time,”“Rate of Infusion,” and “Bubble size Measurements,” the mixing device100 creates, in a matter of milliseconds, a unique non-linear fluiddynamic interaction of the first material 110 and the second material120 with complex, dynamic turbulence providing complex mixing in contactwith an effectively enormous surface area (including those of the deviceand of the exceptionally small gas bubbles of less that 100 nm) thatprovides for the novel electrokinetic effects described herein.Additionally, feature-localized electrokinetic effects (voltage/current)were demonstrated herein (see working Example 20) using a speciallydesigned mixing device comprising insulated rotor and stator features.

As well-recognized in the art, charge redistributions and/or solvatedelectrons are known to be highly unstable in aqueous solution. Accordingto particular aspects, Applicants' electrokinetic effects (e.g., chargeredistributions, including, in particular aspects, solvated electrons)are surprisingly stabilized within the output material (e.g., salinesolutions, ionic solutions). In fact, as described herein, the stabilityof the properties and biological activity of the inventiveelectrokinetic fluids (e.g., RNS-60 or Solas) can be maintained formonths in a gas-tight container, indicating involvement of dissolved gas(e.g., oxygen) in helping to generate and/or maintain, and/or mediatethe properties and activities of the inventive solutions. Significantly,as described in the working Examples herein, the charge redistributionsand/or solvated electrons are stably configured in the inventiveelectrokinetic ionic aqueous fluids in an amount sufficient to provide,upon contact with a living cell (e.g., mammalian cell) by the fluid,modulation of at least one of cellular membrane potential and cellularmembrane conductivity (see, e.g., cellular patch clamp working Examples23 and 24).

As described herein under “Molecular Interactions,” to account for thestability and biological compatibility of the inventive electrokineticfluids (e.g., electrokinetic saline solutions), Applicants have proposedthat interactions between the water molecules and the molecules of thesubstances (e.g., oxygen) dissolved in the water change the collectivestructure of the water and provide for nanoscale cage clusters,including nanostructures comprising oxygen and/or stabilized electronsimparted to the inventive output materials. Without being bound bymechanism, and according to the properties and activities describedherein, the configuration of the nanostructures in particular aspects issuch that they: comprise (at least for formation and/or stability and/orbiological activity) dissolved gas (e.g., oxygen); enable theelectrokinetic fluids (e.g., RNS-60 or Solas saline fluids) to modulate(e.g., impart or receive) charges and/or charge effects upon contactwith a cell membrane or related constituent thereof; and in particularaspects provide for stabilization (e.g., carrying, harboring, trapping)solvated electrons in a biologically-relevant form.

According to particular aspects, and as supported by the presentdisclosure, in ionic or saline (e.g., standard saline, NaCl) solutions,the inventive nanostructures comprise charge stabilized nanostructures(e.g., average diameter less that 100 nm) that may comprise at least onedissolved gas molecule (e.g., oxygen) within a charge-stabilizedhydration shell. According to additional aspects, and as describedelsewhere herein, the charge-stabilized hydration shell may comprise acage or void harboring the at least one dissolved gas molecule (e.g.,oxygen). According to further aspects, by virtue of the provision ofsuitable charge-stabilized hydration shells, the charge-stabilizednanostructure and/or charge-stabilized oxygen containing nano-structuresmay additionally comprise a solvated electron (e.g., stabilized solvatedelectron).

Without being bound by mechanism or particular theory, after the presentpriority date, charge-stabilized microbubbles stabilized by ions inaqueous liquid in equilibrium with ambient (atmospheric) gas have beenproposed (Bunkin et al., Journal of Experimental and TheoreticalPhysics, 104:486-498, 2007; incorporated herein by reference in itsentirety). According to particular aspects of the present invention,Applicants' novel electrokinetic fluids comprise a novel, biologicallyactive form of charge-stabilized oxygen-containing nanostructures, andmay further comprise novel arrays, clusters or associations of suchstructures.

According to the charge-stabilized microbubble model, the short-rangemolecular order of the water structure is destroyed by the presence of agas molecule (e.g., a dissolved gas molecule initially complexed with anonadsorptive ion provides a short-range order defect), providing forcondensation of ionic droplets, wherein the defect is surrounded byfirst and second coordination spheres of water molecules, which arealternately filled by adsorptive ions (e.g., acquisition of a screeningshell of Na⁺ ions to form an electrical double layer) and nonadsorptiveions (e.g., C1 ⁻ ions occupying the second coordination sphere)occupying six and 12 vacancies, respectively, in the coordinationspheres. In under-saturated ionic solutions (e.g., undersaturated salinesolutions), this hydrated ‘nucleus’ remains stable until the first andsecond spheres are filled by six adsorptive and five nonadsorptive ions,respectively, and then undergoes Coulomb explosion creating an internalvoid containing the gas molecule, wherein the adsorptive ions (e.g., Na⁺ions) are adsorbed to the surface of the resulting void, while thenonadsorptive ions (or some portion thereof) diffuse into the solution(Bunkin et al., supra). In this model, the void in the nanostructure isprevented from collapsing by Coulombic repulsion between the ions (e.g.,Na⁺ ions) adsorbed to its surface. The stability of the void-containingnanostructures is postulated to be due to the selective adsorption ofdissolved ions with like charges onto the void/bubble surface anddiffusive equilibrium between the dissolved gas and the gas inside thebubble, where the negative (outward electrostatic pressure exerted bythe resulting electrical double layer provides stable compensation forsurface tension, and the gas pressure inside the bubble is balanced bythe ambient pressure. According to the model, formation of suchmicrobubbles requires an ionic component, and in certain aspectscollision-mediated associations between particles may provide forformation of larger order clusters (arrays) (Id).

The charge-stabilized microbubble model suggests that the particles canbe gas microbubbles, but contemplates only spontaneous formation of suchstructures in ionic solution in equilibrium with ambient air, isuncharacterized and silent as to whether oxygen is capable of formingsuch structures, and is likewise silent as to whether solvated electronsmight be associated and/or stabilized by such structures.

According to particular aspects, the inventive electrokinetic fluidscomprising charge-stabilized nanostructures and/or charge-stabilizedoxygen-containing nanostructures are novel and fundamentally distinctfrom the postulated non-electrokinetic, atmospheric charge-stabilizedmicrobubble structures according to the microbubble model.Significantly, this conclusion is in unavoidable, deriving, at least inpart, from the fact that control saline solutions do not have thebiological properties disclosed herein, whereas Applicants'charge-stabilized nanostructures provide a novel, biologically activeform of charge-stabilized oxygen-containing nanostructures.

According to particular aspects of the present invention, Applicants'novel electrokinetic device and methods provide for novelelectrokinetically-altered fluids comprising significant quantities ofcharge-stabilized nanostructures in excess of any amount that may or maynot spontaneously occur in ionic fluids in equilibrium with air, or inany non-electrokinetically generated fluids. In particular aspects, thecharge-stabilized nanostructures comprise charge-stabilizedoxygen-containing nanostructures. In additional aspects, thecharge-stabilized nanostructures are all, or substantially allcharge-stabilized oxygen-containing nanostructures, or thecharge-stabilized oxygen-containing nanostructures the majorcharge-stabilized gas-containing nanostructure species in theelectrokinetic fluid.

According to yet further aspects, the charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures maycomprise or harbor a solvated electron, and thereby provide a novelstabilized solvated electron carrier. In particular aspects, thecharge-stabilized nanostructures and/or the charge-stabilizedoxygen-containing nanostructures provide a novel type of electride (orinverted electride), which in contrast to conventional solute electrideshaving a single organically coordinated cation, rather have a pluralityof cations stably arrayed about a void or a void containing an oxygenatom, wherein the arrayed sodium ions are coordinated by water hydrationshells, rather than by organic molecules. According to particularaspects, a solvated electron may be accommodated by the hydration shellof water molecules, or preferably accommodated within the nanostructurevoid distributed over all the cations. In certain aspects, the inventivenanostructures provide a novel ‘super electride’ structure in solutionby not only providing for distribution/stabilization of the solvatedelectron over multiple arrayed sodium cations, but also providing forassociation or partial association of the solvated electron with thecaged oxygen molecule(s) in the void—the solvated electron distributingover an array of sodium atoms and at least one oxygen atom. According toparticular aspects, therefore, ‘solvated electrons’ as presentlydisclosed in association with the inventive electrokinetic fluids, maynot be solvated in the traditional model comprising direct hydration bywater molecules. Alternatively, in limited analogy with dried electridesalts, solvated electrons in the inventive electrokinetic fluids may bedistributed over multiple charge-stabilized nanostructures to provide a‘lattice glue’ to stabilize higher order arrays in aqueous solution.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures arecapable of interacting with cellular membranes or constituents thereof,or proteins, etc., to mediate biological activities. In particularaspects, the inventive charge-stabilized nanostructures and/or thecharge-stabilized oxygen-containing nanostructures harboring a solvatedelectron are capable of interacting with cellular membranes orconstituents thereof, or proteins, etc., to mediate biologicalactivities.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures interactwith cellular membranes or constituents thereof, or proteins, etc., as acharge and/or charge effect donor (delivery) and/or as a charge and/orcharge effect recipient to mediate biological activities. In particularaspects, the inventive charge-stabilized nanostructures and/or thecharge-stabilized oxygen-containing nanostructures harboring a solvatedelectron interact with cellular membranes as a charge and/or chargeeffect donor and/or as a charge and/or charge effect recipient tomediate biological activities.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures areconsistent with, and account for the observed stability and biologicalproperties of the inventive electrokinetic fluids, and further provide anovel electride (or inverted electride) that provides for stabilizedsolvated electrons in aqueous ionic solutions (e.g., saline solutions,NaCl, etc.).

In particular aspects, the charge-stabilized oxygen-containingnanostructures substantially comprise, take the form of, or can giverise to, charge-stabilized oxygen-containing nanobubbles. In particularaspects, charge-stabilized oxygen-containing clusters provide forformation of relatively larger arrays of charge-stabilizedoxygen-containing nanostructures, and/or charge-stabilizedoxygen-containing nanobubbles or arrays thereof. In particular aspects,the charge-stabilized oxygen-containing nanostructures can provide forformation of hydrophobic nanobubbles upon contact with a hydrophobicsurface (see elsewhere herein under EXAMPLE 25).

In particular aspects, the charge-stabilized oxygen-containingnanostructures substantially comprise at least one oxygen molecule. Incertain aspects, the charge-stabilized oxygen-containing nanostructuressubstantially comprise at least 1, at least 2, at least 3, at least 4,at least 5, at least 10 at least 15, at least 20, at least 50, at least100, or greater oxygen molecules. In particular aspects,charge-stabilized oxygen-containing nanostructures comprise or give riseto nanobubles (e.g., hydrophobid nanobubbles) of about 20 nm×1.5 nm,comprise about 12 oxygen molecules (e.g., based on the size of an oxygenmolecule (approx 0.3 nm by 0.4 nm), assumption of an ideal gas andapplication of n=PV/RT, where P=1 atm, R=0.082 □057□l.atm/mol.K; T=295K;V=pr² h=4.7×10⁻²² L, where r=10×10⁻⁹ m, h=1.5×10⁻⁹ m, and n=1.95×10⁻²²moles).

In certain aspects, the percentage of oxygen molecules present in thefluid that are in such nanostructures, or arrays thereof, having acharge-stabilized configuration in the ionic aqueous fluid is apercentage amount selected from the group consisting of greater than:0.1%, 1%; 2%; 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%;65%; 70%; 75%; 80%; 85%; 90%; and greater than 95%. Preferably, thispercentage is greater than about 5%, greater than about 10%, greaterthan about 15% f, or greater than about 20%. In additional aspects, thesubstantial size of the charge-stabilized oxygen-containingnanostructures, or arrays thereof, having a charge-stabilizedconfiguration in the ionic aqueous fluid is a size selected from thegroup consisting of less than: 100 nm; 90 nm; 80 nm; 70 nm; 60 nm; 50nm; 40 nm; 30 nm; 20 nm; 10 nm; 5 nm; 4 nm; 3 nm; 2 nm; and 1 nm.Preferably, this size is less than about 50 nm, less than about 40 nm,less than about 30 nm, less than about 20 nm, or less than about 10 nm.

In certain aspects, the inventive electrokinetic fluids comprisesolvated electrons. In further aspects, the inventive electrokineticfluids comprises charge-stabilized nanostructures and/orcharge-stabilized oxygen-containing nanostructures, and/or arraysthereof, which comprise at least one of: solvated electron(s); andunique charge distributions (polar, symmetric, asymmetric chargedistribution). In certain aspects, the charge-stabilized nanostructuresand/or charge-stabilized oxygen-containing nanostructures, and/or arraysthereof, have paramagnetic properties.

By contrast, relative to the inventive electrokinetic fluids, controlpressure pot oxygenated fluids (non-electrokinetic fluids) and the likedo not comprise such charge-stabilized biologically-activenanostructures and/or biologically-active charge-stabilizedoxygen-containing nanostructures and/or arrays thereof, capable ofmodulation of at least one of cellular membrane potential and cellularmembrane conductivity.

In additional aspects, the inventive electrokinetic fluids comprisecharge-stabilized nanostructures as disclosed herein, comprisingvariations in at least one of the ionic components (e.g., variations inthe cation(s) or counterion(s)) and the gas component(s). As describedelsewhere herein, in particular aspects, the inventiveelectrokinetically-altered fluids comprise saline (e.g., one or moredissolved salt(s); e.g., alkali metal based salts (Li+, Na+, K+, Rb+,Cs+, etc.), alkaline earth based salts (e.g., Mg++, Ca++), etc., ortransition metal-based positive ions (e.g., Cr, Fe, Co, Ni, Cu, Zn,etc.,), in each case along with any suitable anion components,including, but not limited to F−, Cl−, Br−, I−, PO₄−, SO₄−, andnitrogen-based anions. Particular aspects comprise mixed salt basedelectrokinetic fluids (e.g., Na+, K+, Ca++, Mg++, transition metalion(s), etc.) in various combinations and concentrations, and optionallywith mixtures of couterions. In particular aspects, the inventiveelectrokinetically-altered fluids comprise standard saline (e.g.,approx. 0.9% NaCl, or about 0.15 M NaCl). In particular aspects, theinventive electrokinetically-altered fluids comprise saline at aconcentration of at least 0.0002 M, at least 0.0003 M, at least 0.001 M,at least 0.005 M, at least 0.01 M, at least 0.015 M, at least 0.1 M, atleast 0.15 M, or at least 0.2 M. In particular aspects, the conductivityof the inventive electrokinetically-altered fluids is at least 10 μS/cm,at least 40 μS/cm, at least 80 μS/cm, at least 100 μS/cm, at least 150μS/cm, at least 200 μS/cm, at least 300 μS/cm, or at least 500 μS/cm, atleast 1 mS/cm, at least 5, mS/cm, 10 mS/cm, at least 40 mS/cm, at least80 mS/cm, at least 100 mS/cm, at least 150 mS/cm, at least 200 mS/cm, atleast 300 mS/cm, or at least 500 mS/cm. In particular aspects, any saltmay be used in preparing the inventive electrokinetically-alteredfluids, provided that they allow for formation of biologically activesalt-stabilized nanostructures (e.g., salt-stabilized oxygen-containingnanostructures) as disclosed herein.

According to particular aspects, the biological effects of the inventivefluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the ionic components of the fluids, and/or by alteringthe gas component of the fluid.

According to particular aspects, the biological effects of the inventivefluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the gas component of the fluid. In preferred aspects,oxygen is used in preparing the inventive electrokinetic fluids. Inadditional aspects mixtures of oxygen along with at least one other gasselected from Nitrogen, Oxygen, Argon, Carbon dioxide, Neon, Helium,krypton, hydrogen and Xenon. As described above, the ionic componentss(cation(s) and and/or counterion(s)) may also be varied, including alongwith varying the gas constitutent(s).

Systems for Making Gas-Enriched Fluids

The presently disclosed system and methods allow gas (e.g. oxygen) to beenriched stably at a high concentration with minimal passive loss. Thissystem and methods can be effectively used to enrich a wide variety ofgases at heightened percentages into a wide variety of fluids. By way ofexample only, dissolved oxygen can achieve levels of dissolved oxygenranging from at least about 5 ppm, at least about 10 ppm, at least about15 ppm, at least about 20 ppm, at least about 25 ppm, at least about 30ppm, at least about 35 ppm, at least about 40 ppm, at least about 45ppm, at least about 50 ppm, at least about 55 ppm, at least about 60ppm, at least about 65 ppm, at least about 70 ppm, at least about 75ppm, at least about 80 ppm, at least about 85 ppm, at least about 90ppm, at least about 95 ppm, at least about 100 ppm, or any value greateror therebetween using the disclosed systems and/or methods. Inaccordance with a particular exemplary embodiment, oxygen-enriched watermay be generated with levels of about 30-60 ppm of dissolved oxygen.

Table 4 illustrates various partial pressure measurements taken in ahealing wound treated with an oxygen-enriched saline solution (Table 4)and in samples of the gas-enriched oxygen-enriched saline solution ofthe present invention.

TABLE 4 TISSUE OXYGEN MEASUREMENTS Probe Z082BO In air: 171 mmHg 23° C.Column Partial Pressure (mmHg) B1 32-36 B2 169-200 B3  20-180* B4 40-60*wound depth minimal, majority >150, occasional 20 s

Rayleigh Effects

If a strong beam of light is passed through a transparent gaseous orliquid medium containing solid or liquid particles, or even molecules ofextremely high molecular weight, the light is scattered away from thedirection of its incident path. The scattering is due to theinterference effects that arise from the density fluctuations in thescattering medium (i.e. the presence of particles or very high molecularweight molecules.) There are two types of light scattering. The firstinvolves the wavelength of the scattered light differing from that ofthe incident light and is called Raman scattering. The other typescattering involves when the scattered light has the same wavelength ofthe incident light and is called Rayleigh scattering. In Rayleighscattering, the intensity of the scattered light is proportional to theproduct of the intensity of the incident light and the attenuationconstant, a function of the refractive index and the Rayleigh constant.The Rayleigh constant is a somewhat involved function of the molecularweight of the scattering substance and thus a measurement of theintensity of the scattered light can give a value for the molecularweight. This scattering phenomenon is used in a number of liquidchromatography detectors.

Water processed through the mixing device 100 has been demonstrated tohave detectible structural differences when compared with normalunprocessed water. For example, processed water has been shown to havemore Rayleigh scattering than is observed in unprocessed water. In theexperiments that were conducted, samples of processed and unprocessedwater were prepared (by sealing each in a separate bottle), coded (forlater identification of the processed sample and unprocessed sample),and sent to an independent testing laboratory for analysis. Only afterthe tests were completed were the codes interpreted to reveal whichsample had been processed by the mixing device 100.

At the laboratory, the two samples were placed in a laser beam having awavelength of 633 nanometers. The fluid had been sealed in glass bottlesfor approximately one week before testing. With respect to the processedsample, Sample B scattered light regardless of its position relative tothe laser source. However, “Sample A” did not. After two to three hoursfollowing the opening of the bottle, the scattering effect of Sample Bdisappeared. These results imply the water exhibited a memory causingthe water to retain its properties and dissipate over time. Theseresults also imply the structure of the processed water is opticallydifferent from the structure of the unprocessed fluid. Finally, theseresults imply the optical effect is not directly related to DO levelsbecause the DO level at the start was 45 ppm and at the end of theexperiment was estimated to be approximately 32 ppm.

Generation of Solvated Electrons

Additional evidence indicates that the mixing occurring inside themixing device 100 generates solvated electrons within the outputmaterial 102. This conclusion results from conditions observed withrespect to the dissolved oxygen probe effects used in measuring the DOlevels within various processed solutions. Due to the experiences viewedwith respect to the polarographic dissolved oxygen probes, it is abelief that the processed fluid exhibits an electron capture effect andthus the fluid includes solvated electrons.

There are two fundamental techniques for measuring dissolved oxygen(“DO”) levels electrically: galvanic measuring techniques andpolarographic measurements. In both techniques, the DO level sensorincludes two electrodes, an anode and a cathode, which are both immersedin electrolyte within the sensor body. An oxygen permeable membraneseparates the anode and cathode from the solution being tested. Thecathode is a hydrogen electrode and carries negative potential withrespect to the anode. The electrolyte solution surrounds the electrodepair and is contained by the membrane. With no oxygen, the cathodebecomes polarized with hydrogen and resists the flow of current. Whenoxygen passes through the membrane, the cathode is depolarized andelectrons are consumed. In other words, oxygen diffuses across themembrane and interacts with the internal components of the probe toproduce an electrical current. The cathode electrochemically reduces theoxygen to hydroxyl ions according to the following equation:

O₂+2H₂O+4E⁻=4OH⁻

When attempting to measure DO levels in a solution processed by themixing device 100, an overflow condition has been repeatedly experiencedwherein the dissolved oxygen meter actually displays a reading that ishigher than the meter is capable of reading. Independent means, aWinkler Titration, reveals a much lower DO level for the solution thanindicated by the probe. Typically, in a device such as the Orion 862,having a maximum reading of 60 ppm, the meter will overflow and have thehigh oxygen level indication if left in bulk processed water for severalminutes.

Because the overload is not caused by dissolved oxygen in the fluid, itis believed solvated electrons must be causing the overload. In otherwords, solvated electrons are accompanying the processed water acrossthe membrane. These electrons are attracted to the anode and cause thecurrent observed. It is a further belief that these electrons arecaptured in a cage or cluster mechanism within the solution.

Compositions Comprising Forms of Hydrated (Solvated) Electrons Impartedto the Inventive Compositions by the Inventive Processes

In certain embodiments as described herein (see under “Double-layer”),the gas-enriched fluid is generated by the disclosed electromechanicalprocesses in which molecular oxygen is diffused or mixed into the fluidand may operate to stabilize charges (e.g., hydrated (solvated)electrons) imparted to the fluid. Without being bound by theory ormechanism, certain embodiments of the present invention relate to aoxygen-enriched fluid (output material) comprising charges (e.g.,hydrated (solvated) electrons) that are added to the materials as thefirst material is mixed with oxygen in the inventive mixer device toprovide the combined output material. According to particular aspects,these hydrated (solvated) electrons (alternately referred to herein as‘solvated electrons’) are stabilized in the inventive solutions asevidenced by the persistence of assayable effects mediated by thesehydrated (solvated) electrons. Certain embodiments may relate tohydrated (solvated) electrons and/or water-electron structures,clusters, etc., (See, for example, Lee and Lee, Bull. Kor. Chem. Soc.2003, v. 24, 6; 802-804; 2003).

Horseradish peroxidase (HRP) effects. Horseradish peroxidase (HRP) isisolated from horseradish roots (Amoracia rusticana) and belongs to theferroprotoporphyrin group (Heme group) of peroxidases. HRP readilycombines with hydrogen peroxide or other hydrogen donors to oxidize thepyrogallol substrate. Additionally, as recognized in the art, HRPfacilitates auto-oxidative degradation of indole-3-acetic acid in theabsence of hydrogen peroxide (see, e.g., Heme Peroxidases, H. BrianDunford, Wiley-VCH, 1999, Chapter 6, pages 112-123, describing thatauto-oxidation involves a highly efficient branched-chain mechanism;incorporated herein by reference in its entirety). The HRP reaction canbe measured in enzymatic activity units, in which Specific activity isexpressed in terms of pyrogallol units. One pyrogallol unit will form1.0 mg purpurogallin from pyrogallol in 20 sec at pH 6.0 at 20° C. Thispurpurogallin (20 sec) unit is equivalent to approx. 18 μM units per minat 25° C.

It is known that Horseradish peroxidase enzyme catalyzes theauto-oxidation of pyrogallol by way of facilitating reaction with themolecular oxygen in a fluid. (Khajehpour et al., PROTEINS: Struct,Funct, Genet. 53: 656-666 (2003)). It is also known that oxygen bindsthe heme pocket of horseradish peroxidase enzyme through a hydrophobicpore region of the enzyme (between Phe68 and Phe142), whose conformationlikely determines the accessibility of oxygen to the interior. Accordingto particular aspects, and without being bound by mechanism, becausesurface charges on proteins are known in the protein art to influenceprotein structure, the solvated electrons present in the inventivegas-enriched fluid may act to alter the conformation of the horseradishperoxidase such that greater oxygen accessibility may result. Thegreater accessibility of oxygen to the prosthetic heme pocket of thehorseradish peroxidase enzyme may in turn allow for increased HRPreactivity, when compared with prior art oxygenated fluids(pressure-pot, fine-bubbled).

In any event, according to particular aspects, production of outputmaterial using the inventive methods and devices comprises a processinvolving: an interfacial double layer that provides a charge gradient;movement of the materials relative to surfaces pulling charge (e.g.,electrons) away from the surface by virtue of a triboelectric effect,wherein the flow of material produces a flow of solvated electrons.Moreover, according to additional aspects, and without being bound bymechanism, the orbital structure of diatomic oxygen creates chargeimbalances (e.g., the two unpaired electrons affecting the hydrogenbonding of the water) in the hydrogen bonding arrangement within thefluid material (water), wherein electrons are solvated and stabilizedwithin the imbalances.

Several chemical tests of the inventive oxygen-enriched fluid for thepresence of hydrogen peroxide were conducted as described below, andnone of these tests were positive (sensitivity of 0.1 ppm hydrogenperoxide). Thus, the inventive oxygen-enriched fluid of the instantapplication contain no, or less than 0.1 ppm hydrogen peroxide.

According to particular aspects, despite the absence of hydrogenperoxide, the inventive combination of oxygen-enrichment and solvatedelectrons imparted by the double-layer effects and configuration of thepresently claimed devices may act to alter the conformation and/or hemegroup accessibility of the horseradish peroxidase.

Glutathione Peroxidase Study

The inventive oxygen-enriched output fluid material was tested for thepresence of hydrogen peroxide by testing the reactivity with glutathioneperoxidase using a standard assay (Sigma). Briefly, glutathioneperoxidase enzyme cocktail was constituted in deionized water and theappropriate buffers. Water samples were tested by adding the enzymecocktail and inverting. Continuous spectrophotometric rate determinationwas made at A₃₄₀ nm, and room temperature (25 degrees Celsius). Samplestested were: 1. deionized water (negative control), 2. inventiveoxygen-enriched fluid at low concentration, 3. inventive oxygen-enrichedfluid at high concentration, 4. hydrogen peroxide (positive control).The hydrogen peroxide positive control showed a strong reactivity, whilenone of the other fluids tested reacted with the glutathione.

Bioreactor Systems Comprising the Inventive Mixing Devices

Producing significant quantities of target products, such as proteins,polypeptides, nucleic acids, therapeutic agents, and other products inhost cell systems are possible due to advances in molecular biology. Forexample, recombinant proteins are produced in a host cell systems bytransfecting the host cell with nucleic acids (e.g. DNA) encoding aprotein of interest. Next, the host cell is grown under conditions whichallow for expression of the recombinant protein. Certain host cellsystems can be used to produce large quantities of recombinant proteinswhich would be too impractical to produce by other means.

In addition, enzymatic and/or reaction fermentations, with or withouthost cells, are utilized for example in producing foodstuff andbeverages, in treating wastewater, or in environmental cleanup.

Cell culturing processes, or cellular fermentation, typically useprokaryotic or eukaryotic host cells to produce recombinant proteins.The fermentation is typically conducted in physical containers (e.g.stirred tanks) called fermentors or tank bioreactors. The fermentationprocess itself may comprise (1) discontinuous operation (batch process),(2) continuous operation, or (3) semi-continuous operations (such as thefed-batch process), or any combination of these.

Since the aim of large scale production of pharmaceutical drugs (e.g.biologicals) or other target products is to provide improvedmanufacturing processes and reduced costs, there is a need for improvedbioreactor equipment, methods, and media for preparation of these targetproducts.

The present disclosure sets forth novel gas-enriched fluids, including,but not limited to gas-enriched water, saline solutions (e.g., standardaqueous saline solutions), cell culture media, as well as novel methodsand biological and chemical reactor systems for use in these applicationprocesses, and others.

Certain embodiments disclosed herein relate to systems, media, andmethods for producing a target product, such as a protein.

In certain embodiments, a target product may refer to a protein,peptide, polypeptide, nucleic acid, carbohydrate, polymer, micelle, andany mixture thereof.

The target product is typically produced by a vehicle, such as a hostcell, which is associated with the gas-enriched fluid in a chemical orbiological reactor. Reactors may include standard reactors, such ascontinuous feed, discontinuous feed, and/or semi-continuous feed.Reactors may also include a cell culture vessel (such as a plate, flask,or tank), a plant, an animal, a fungus, an algae, or other organism. Forexample, a plant that is associated with the gas-enriched fluid of thepresent invention may comprise plant cells acting as vehicles that aidin the production of the target product (for example, naturallyoccurring plant matter or genetically altered plant matter).

In certain embodiments, the vehicles utilized with the gas-enrichedfluids or solutions (including media) may include prokaryotic cells oreukaryotic cells. More specifically, the living cells may includebacterial (e.g. E. coli, Salmonella, Streptococcus, Staphylococcus,Neisseria, Nocardia, Mycoplasma, etc.), fungal (e.g. yeasts, molds,mushrooms, etc.), plant (tobacco, maize, soybean, fruit or vegetable,etc.), animal (mammalian, insect, etc.) archebacterial (blue greenalgae), protist, human embryonic kidney (HEK) cells, HeLa cells,hybridoma cells, Madin-Darby Canine Kidney (MDCK) cells, stem cells,cell lines (including SP2/0 and NSO), African Green Monkey Kidney (Vero)cells, Spodoptera frugiperda (army worm), Trichoplusia ni (cabbagelooper), and other cells. In addition, viruses (such as bacteriophage,baculovirus, vaccinia, and other viruses) may be employed in thebioreactors of the present invention.

The bioreactor may comprise an airlift reactor, a packed bed reactor, afibrous bed reactor, a membrane reactor, a two-chamber reactor, astirred-tank reactor, a hollow-fiber reactor, or other reactor designedto support suspended or immobilized cell growth.

In cases of recombinant or target protein production, a balanced batchand/or feed medium must encourage optimal cell growth and expression ofthe recombinant protein. The medium, or media, is termed “minimal” if itonly contains the nutrients essential for growth. For prokaryotic hostcells, the minimal media typically includes a source of carbon,nitrogen, phosphorus, magnesium, and trace amounts of iron and calcium.(Gunsalus and Stanter, The Bacteria, V. 1, Ch. 1 Acad. Press Inc., N.Y.(1960)). Most minimal media use glucose as a carbon source, ammonia as anitrogen source, and orthophosphate (e.g. PO₄) as the phosphorus source.The media components can be varied or supplemented according to thespecific prokaryotic organism(s) grown, in order to encourage optimalgrowth without inhibiting target protein production. (Thompson et al.,Biotech. and Bioeng. 27: 818-824 (1985)). This allows for higher levelsof production with lower cost.

In addition to the chemical composition of the media, other factors mayaffect cell growth and/or target protein production. These factorsinclude, but are not limited to pH, time, cultivation temperature,amount of dissolved oxygen or other gas(es), and partial pressure ofthose dissolved gasses. During the fermentation process, the pH of themedia is typically altered due to the consumption of ammonia, ormicroorganism synthesis of certain metabolic products, e.g., acetic acidand lactic acid. Since altered pH may be unfavorable for optimal cellgrowth, it may be necessary or desirable to maintain the medium at acertain pH (i.e. by addition of acids or bases). The pH and otherprocess parameters can be monitored manually or by automatic devices.

Inventive Gas-Enriched Fluids

Enriching a fluid with another fluid may result in a solution orsuspension of the two fluids, depending on the physical and chemicalproperties of the two fluids. In particular, enriching a liquid with agas (e.g. oxygen) may be beneficial for certain applications, includingtherapeutic treatments. As utilized herein, “fluid,” may generally referto a liquid, a gas, a vapor, a mixture of liquids and/or gases, a liquidand/or gas solution, or any combination thereof, for any particulardisclosed embodiment. Furthermore, in certain embodiments a “liquid” maygenerally refer to a pure liquid or may refer to a gel, sol, emulsion,fluid, colloid, dispersion, suspension, or mixture, as well as anycombination thereof; any of which may vary in viscosity.

In particular embodiments, the dissolved gas comprises oxygen. In otherparticular embodiments, the dissolved gas comprises nitrogen, carbondioxide, carbon monoxide, ozone, sulfur gas, nitrous oxide, nitricoxide, argon, liquefied petroleum gas, helium, natural gas, or others.

One particular advantage of embodiments of the present invention relatesto the gas-enriched fluids' long-term diffused gas (particularly oxygen)levels, which allows for long-term bio-availability of the gas tocellular or chemical reactors. The long-term bio-availability of gassesin the gas-enriched fluids of the present invention allow for increasedtarget product production and/or improved enzymatic or other chemicalreactions that benefit from the gas-enriched fluids (includingoxygenated media) of the present invention.

In some instances, living cells may be grown in a bioreactor orfermentation chamber in order to promote cell growth and/or productionof the intended target product. While some living cells require amixture of gasses in order to sustain or promote their survival orpropagation, cell growth may be hindered or ceased if a particular gas,such as oxygen, is present at too high of a concentration.

For example, mammalian cells, such as Chinese Hamster Ovary (CHO) cells,require oxygen in order to proliferate. However, the existing techniquesin the art for diffusing gasses, such as oxygen, into the bioreactorfluids have a detrimental effect on mammalian cell cultures. Forexample, the cells may be destroyed or rendered non-viable in instanceswhere the diffused gas bubbles rupture or coalesce within the culturemedia, which is particularly common at a gas-to-liquid interface.Accordingly, the present invention represents an advance that would nothave occurred in the ordinary course since the existing knowledge in theart teaches that the levels of dissolved gas, particularly the levels ofdissolved oxygen, in the gas-enriched media disclosed herein ispredicted to be harmful or detrimental. However, the gas-enriched fluidmedia as described herein result in imparting at least one beneficialadvantage to cell cultures selected from the group consisting of:enhanced cell growth (e.g. rate and/or number) increased target productyield (e.g. amount), increased rate of target product production,improved vehicle cell viability, increased efficiency of target productproduction, increased ease in target product purification, and the like.In certain embodiments, one or more of these beneficial advantages areconveyed to cell cultures without proving injurious to the cellsthemselves.

In other embodiments, an a cellular reaction may utilize thegas-enriched fluids and methods of the present invention, includinggeneral chemical and/or enzymatic reactions. Examples of such reactionsinclude, but are not limited to, wastewater treatment, purification ofwater (such as treating municipal water, home drinking purifiers,cleaning swimming pools or aquariums, etc.), homogenization of milk,hydrogenation of oils, gas-enriching fuels, and others.

In further embodiments, the gas-enriched fluid maintains a dissolved gasenrichment level of at least 10 ppm, at least 15 ppm, at least 20 ppm,at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, atleast 45 ppm, at least 50 ppm, at least 55 ppm, at least 60 ppm, atleast 65 ppm, at least 70 ppm, at least 75 ppm, at least 80 ppm, atleast 85 ppm, at least 90 ppm, at least 100 ppm, or any value greater ortherebetween, at atmospheric pressure. In certain instances, thegas-enriched fluid maintains its dissolved gas enrichment level (i.e.the level of the gas enriched in the fluid) for a period of at least 10days, at least 20 days, at least 30 days, at least 40 days, at least 50days, at least 60 days, at least 70 days, at least 80 days, at least 90days, at least 100 days, at least 110 days, at least 120 days, at least130 days, or greater or any value therebetween, within a sealedcontainer at atmospheric pressure.

In one particular embodiment, the host material comprises water or watervapor. In another particular embodiment, the host material comprisesother fluids (i.e. gasses or liquids) such as wastewater, toxicmaterials, potable water, milk, juice, yogurt, soft drinks (particularlycarbonated beverages), ethanol, methanol, polymers (such as plastic orrubber compounds), oil (edible or non-edible), emulsions, suspensions,aqueous carriers, non-aqueous carriers, and the like.

In certain embodiments, multiple gasses may be used to enrich or infusea host fluid. In certain embodiments, ozone and/or oxygen may be used tobreak down complex structures into smaller substructures, particularlyif used with sonochemistry techniques, as described herein inter alia.

In certain embodiments, the gas-enriched fluid or other host material ofthe present invention has characteristics that may be more similar tothe gas that has enriched the fluid or other host material, or it mayhave characteristics that are more similar to the fluid (e.g. gas orliquid) or other host material itself.

In certain embodiments, a gas-enriched fluid or solution comprisesgas-enriched culture media. In particular embodiments, the gas-enrichedmedia comprises oxygenated or oxygen-enriched media. In certainembodiments, the gas-enriched fluid or gas-enriched host material mayinclude further processing, such as by filtering, separating, modifyingor altering various constituents of the fluid or host material.

Packaged Gas-Enriched Fluids

Certain embodiments disclosed herein relate to gas-enriched fluids thathave high levels of dissolved or diffused gases (particularly oxygen)that may be produced by various methods, including those describedherein. In certain embodiments, the gas-enriched fluid may be producedin a biomass production facility and applied directly to a bioreactorsystem. Alternatively, the gas-enriched fluid may be packaged anddistributed for use at other locations. In the event that thegas-enriched fluid is packaged, such packaging may include a sterilepackage such as a glass or plastic container, flexible foil or plasticpouches, sealed boxes (particularly waxed boxes), and the like. In thecase of sealed packages, the gas-enriched fluid may maintain a highlevel of dissolved or diffused gas for several days, several weeks, orseveral months. In certain embodiments, the sealed container (i.e.enclosed with a cap, cover or other enclosure that is at leastsemi-impermeable to gas exchange) maintains the diffused nature of thefluid at least 2 weeks, at least 4 weeks, at least 2 months, at least 4months, at least 6 months, at least 8 months, at least 10 months, atleast 12 months, or any value greater or therebetween.

Gas-Enriched Fluids in Biological or Chemical Reactors

As illustrated in FIGS. 122 and 123, a biological or chemical reactorsystem 3300 a may be used for conventional large-scale cell-culturing orchemical processing to achieve the production of the target product3318. The target product 3318 may include, but not be limited to,proteins, antibodies, synthetic peptides, active pharmaceutical agents,foodstuff or beverage products (such as wine; beer; soft drinks; fruitor vegetable juices); plant products (flowers, cotton, tobacco, wood,paper, wood or paper pulp, etc.); ethanol, methanol, paints, fruit orvegetables or fruit or vegetable products such as jellies, jams, sauces,pastes, and the like; cheese or cheese products; nuts or nut products(such as peanut butter, almond paste, etc.); meat or meat products,grain flours or products including bread, cereal, pasta, and the like;slurries or mixtures of any of these, processed polymers (includingplastics, and other polymers), petroleum products, and others.

In certain embodiments, in the case of using a vessel reactor, such as atank reactor, the target product resides within inclusion bodies,particularly when E. coli cells are utilized. The target product may beobtained by processing the inclusion bodies, for example by usinghigh-pressure homogenizers or other techniques.

In particular embodiments in which the reactor is a biological reactorsystem, the system 3306 includes a source 3308 of culture cells 3310 tobe cultured, a source 3302 of culture media 3304, a biological reactor3306, and a harvesting and purification system 3316, for producing thetarget product 3318. The culture cells 3310 are geneticallypredetermined to produce proteins or the like that constitute the targetproduct 3318, and the culture medium 3304 may comprise a sterile mediumof a type that provides nourishment for the proliferation of culturecells 3310. In this particular exemplary embodiment, the sterile medium3304 is introduced into the internal chamber (which may be referred toas the “fermentation chamber”) of the reactor 3306 from the source 3302.From the source 3308, the culture cells 3310 are provided such that thecells 3310 and medium 3304 are combined into a broth 3312 in thefermentation chamber of the bioreactor 3306.

The appropriate base medium 3304 to be utilized in the reactor system3300 a may be formulated to provide optimal nourishment and growth tothe cell culture 3310. Medium 3304 is preferably a fluid (e.g. liquid orgas) medium, more preferably a liquid medium or a solid-liquid mediumthat is selected based on the certain variables, such as thecharacteristics and objectives of the overall bioreactor system 3300 a,the cost, the type of cells being cultured from the cell culture 3310,the desired production parameters, the type of culturing and mediamanagement process used in the reactor 3306, the type of downstreamharvest and purification processes 3316, and the target activepharmaceutical ingredient 3318. Various cell culture media presentlyused may be adapted for use or gas-enrichment by the present invention.

In certain embodiments, a suitable base medium 3304 may include but notbe limited to a serum-supplemented medium, a hydrolysate medium,chemically-synthesized medium, chemically-defined medium, a serum-freemedium, any combination of these or other media.

In certain embodiments, the gas-enriched media may be supplemented withtransferrins, albumins, fetuins, protein hydrolysates, or otheradditives, preservatives, nutrients, fillers, shear protectants (such asPluronic F68), or active or inactive agents.

In addition, the medium may be formulated for cells that are attached tosome type of support within the bioreactor 3306, rather than suspendedin the broth 3312. In all embodiments that utilize a medium 3304, themedium 3304 is formulated to meet the nutritional requirements of theindividual cell type in the cell culture 3310, and typically compriseminerals, salts, and sugars.

In certain embodiments, medium 3304 and/or broth 3312 are gas-enrichedusing the presently disclosed mixing devices 100, in order to dissolveor diffuse gases (such as oxygen) into, for example, the media, both orcomponents thereof, in concentrations of at least about 8 ppm, at leastabout 10 ppm, at least about 20 ppm, at least about 25 ppm, at leastabout 30 ppm, at least about 35 ppm, at least about 40 ppm, at leastabout 50 ppm, at least about 60 ppm, at least about 70 ppm, at leastabout 80 ppm, at least about 90 ppm, at least about 100 ppm, or anyvalue greater or therebetween. In certain embodiments, the gas-enrichedmedium and/or broth contains less than about 160 ppm.

In certain embodiments, the typical biological or chemical reactor isloaded with sterilized raw materials (nutrients, reactants, etc.) alongwith air or specific gas, as well as cells for a biological reactor.Other agents may be added to the mixture, including anti-foamingchemicals or agents, pH controlling agents, and/or other agents. Thetarget product is typically recovered by separating the cells, and/ordisrupting the cells in order to extract the product, concentrating theproduct, and purifying, drying, or further processing the product.

Many different types of bioreactor systems are in use today, any ofwhich can be used with the gas-enriched media of the present invention.For example, air-lift bioreactors are commonly used with bacteria, yeastand other fungi; fluidized-bed bioreactors are commonly used withimmobilized bacteria, yeast and other fungi, as well as activatedsludge; microcarrier bioreactors are commonly used with mammalian cellsimmobilized on solid particles; surface tissue propagators are commonlyused with mammalian cells, tissue grown on solid surfaces, and tissueengineering; membrane bioreactors, hollow fibers and roto-fermentors aretypically used with bacteria, yeast, mammalian cells, and plant cells;modified stir-tank bioreactors are commonly used with immobilizedbacteria, yeast, and plant cells; modified packed-bed bioreactors arecommonly used with immobilized bacteria, yeast, and other fungi; towerand loop bioreactors are commonly used with bacteria and yeast; vacuumand cyclone bioreactors are commonly used with bacteria, yeast, andfungi; and photochemical bioreactors are commonly used withphotosynthetic bacteria, algae, cyanobacteria, plant cell culture,and/or DNA plant cells.

Since living cells, including bacteria, yeast, plant cells, mammaliancells, and fungal cells require molecular oxygen as an electron acceptorin the bioxidation of substrates (such as sugars, fats, and proteins),cell culture media that is highly oxygenated is beneficial to the livingcells. In a standard oxidation-reduction reaction, glucose is oxidizedto make carbon dioxide, while oxygen is reduced to make water. Molecularoxygen accepts all of the electrons released from the substrates duringaerobic metabolism. Thus, in order to provide the maximum amount ofbio-available oxygen to the growing cells, it is necessary to ensurethat the oxygen transfer from the air bubbles (gas phase) to the liquidphase occurs quickly. When no oxygen accumulates in the liquid phase,the rate of the oxygen transfer is the same as the rate of the oxygenuptake by the growing cells.

The oxygen requirements of microorganisms is defined as a standardformula, that is in units of QO₂. Where QO₂ is the mass of oxygenconsumed divided by the unit weight of dry biomass cells in thebioreactor multiplied by time. Conversely, the rate of accumulation ofoxygen is equal to the net rate of oxygen supply from air bubbles minusthe rate of oxygen consumption by cells.

In addition to a multitude of bioreactor types, each bioreactor mayutilize a particular impeller type or types, such as marine-typepropellers, flat-blade turbines, disk flat-blade turbines, curved-bladeturbines, pitched-blade turbines, paddles, and shrouded turbines. Theimpeller or turbine may create a vortex pattern of mixing in thebioreactor, or a uniform mixing.

In certain embodiments, the gas-enriched fluid of the present inventionrelates to a sustained bio-availability of the gas such that a gradualrelease of the gas occurs over time. This gradual or “time” release isbeneficial to the vehicles, such as cultured cells, particularly whenthe gas released from the gas-enriched fluid comprises oxygen. Thus,fermentation, or the biochemical synthesis of organic compounds by cells3310, typically involve a relatively fast growth phase, facilitated bythe concentrations of diffused or dissolved gas in the broth 3312, aswell as by temperature control and by mixing the medium 3304 and thecell culture 3310 in the fermentation chamber of the bioreactor 3306.Particular exemplary embodiments are depicted in the figures, but mayinclude additional components or tanks. Mixing may be enhanced byrotating vanes or the like within bioreactor 3306, and by reintroductionof fresh and/or freshly re-diffused supplies of medium 3304 from any ofthe lines 3332, 3338 or 3334, as described herein inter alia.

In one particular exemplary embodiment depicted in FIG. 122, theenrichment processing of the medium and/or broth to introduce the gas(e.g. oxygen) in a cell culture medium may occur at various points inthe system. For example, the medium may be gas-enriched prior tointroducing the medium 3304 into the system 3300 a at source 3302, orafter such introduction at one or more locations “A,” “B,” “C,” “D,” “E”or combinations thereof. Gas-enriched fluid that may be introduced atthe source 3302, whether enriched at the site of the bioreactor or at aseparate location. If the gas-enriched fluid is enriched at a separatelocation, it may be transported to the source 3302 in appropriatecontainers or plumbing.

In certain embodiments, each of the locations “A,” “B,” and “C,” of FIG.122 represent alternative locations for introduction of a gas-enrichmentdiffuser device 1—within the bioreactor system 3300A. In the event thatthe introduction occurs at point “A,” the flow of medium from tank 3304through the upper section of 3332A of 3332, the medium may be directedthrough the gas-enrichment mixer/diffuser device 100 located at position“A,” and medium 3304 with dissolved gases therein proceeds from themixer/diffuser device 100 through 3332B and into the fermentationchamber of the bioreactor 3306.

With reference to FIG. 123, the medium 3304 from tank 3302 may bedirected through line 3332A into a pump 3410 and, subsequently into thehost material input of the gas-enrichment diffuser device 100. The pump3410, is preferably a variable speed pump, which may be controlled by acontroller 3390, based, in part, on pressure readings detected bypressure sensor 3415. While certain embodiments will utilize manualgauges as pressure detectors, from which an operator may manually adjustthe speed of the pump 3410, and other components of the system 3300 a or3300 b, controller 3390 preferably receives an electrical signal fromsensor 3415 such that controller 3390 will automatically adjust thespeed of pump 3410. As will be evident from further descriptions herein,the speed of pump 3410 may also be based on algorithms within thecontroller 3390 which depend, in part, on the state of other componentsof the system 3400 (such as valves 3420, 3421 and sensors 3425).

Alternatively, the gas-enrichment mixer/diffuser device 100 may bepositioned at location “B” such that the medium 3304 is processedtogether with medium 3310. In this particular case, cells 3310 andmedium 3304 are mixed in flow using a conventional mixing nozzle andsubsequently introduced into the mixer/diffuser device 100, wherebeneficial gases are infused into the mixed liquid of medium 3304 andcells 3310. The resulting gas-enriched medium is then directed into thefermentation tank of the bioreactor 3306.

As shown in FIG. 122, cells 3310 may be combined with medium 3304, andfollowing fermentation and/or development of the target product, thecontents of the bioreactor 3306 may then be directed through line 3336to a harvesting and purification stage. Once purified, the targetproduct is directed through line 3339 to a target production tank 3318.

With reference to FIG. 123, in certain embodiments, the gas-enrichmentmixer/diffuser device 100 combines the flow of a medium 3304 with a flowof a gas from line 3426. Preferably, the gas to be combined with medium3304 flows from an oxygen tank 3450 and is metered by a valve 3420,which is controlled by controller 3390.

In certain embodiments, the gas-enrichment mixer/diffuser device 100 isdirected through line 3332 b directly into the fermentation tank by areactor 3306. Alternatively, the gas-enriched fluid may be directedthrough line 3332 b to another blending.

With reference to FIG. 122, a bioreactor system 3300 a, may include anadditional system (such as a perfusion system) 3314 that beginsprocessing the broth from the bioreactor 3306. During the perfusionprocess 3314, the medium 3304 is continuously added to the broth 3312 tonourish the cell culture 3310, which is then mixed throughout the broth3312. Simultaneously, cell or other waste may be continuously removedfrom the broth 3312, typically at the same rate as new medium 3304 isadded. As indicated herein above, gas-enrichment may also occur atpositions “D” or “E,” or at both positions “D” and “E.”

The perfusion system can allow for removal of cellular waste and debris,as well as the target product, while retaining the cells in thebioreactor 3306. The perfusion system thus reduces waste accumulationand nutrient fluctuations, thereby allowing for higher cell density andproductivity. Retention of the cells in the bioreactor may be achievedthrough various methods, including centrifugation, internal or externalspin filters, hollow fiber modules, cross-flow filtration, depthfiltration, any combination of these or other means. In otherembodiments, the accumulation of waste products may be regulated by useof a glutamine synthetase expression system.

With reference to FIG. 124, particular exemplary embodiments utilizemultiple gas sources 3502 and 3504 as shown, such that the nature of thegas being diffused into the broth 3312 can be changed depending on thestage of fermentation within the bioreactor 3306. Hence, in a preferredembodiment, the cell culture medium is enriched with oxygen during theproliferative phase of fermentation. Subsequently, carbon dioxide,nitrous oxide, or another gas may be substituted to facilitate otherstages of the fermentation process, particularly with processes thatvary from aerobic to anaerobic.

The bioreactor may comprise an airlift reactor, a packed bed reactor, afibrous bed reactor, a membrane reactor, a two-chamber reactor, astirred-tank reactor, a hollow-fiber reactor, or other reactor designedto support suspended or immobilized cell growth.

In one particular embodiment, the bioreactor 3306 is a continuousstirred-tank reactor, comprising heat exchange and refrigerationcapabilities, sensors, controllers, and/or a control system to monitorand control the environmental conditions within the fermentationchamber. Monitored and controlled conditions may include gas (e.g. air,oxygen, nitrogen, carbon dioxide, nitrous oxide, nitric oxide, sulfurgas, carbon monoxide, hydrogen, argon, helium, flow rates, temperature,pH, dissolved oxygen levels, agitation speed, circulation rate, andothers. Additionally, the bioreactor 3306 may further compriseCleaning-in-Place (CIP) or Sterilization-in-Place (SIP) systems, whichmay be cleaned and/or sterilized without assembly or disassembly of theunits.

In one particular embodiment, the bioreactor 3306 performs a continuousfermentation cycle, continuously adding medium 3304 to the fermentationsystem with a balancing withdrawal, or harvest, of the broth 3312 fortarget product extraction.

In alternate embodiments, the bioreactor 3306 may perform batchfermentation cycles, fed-batch fermentation cycles, or fed-batchfermentation cycles with the gas-enriched fluids. Typically, batchfermentation cycles—in which all of the reactants are loadedsimultaneously—are used for small scale operations or for themanufacture of expensive products or for processes that may be difficultto convert into continuous operations. In a typical process, the brothis fermented for a defined period to completion, without furtheradditions of the medium. The concentration varies with time, but istypically uniform at any one particular time point. Agitation serves tomix separate feed lines as well as enhance heat transfer.

For batch fermentation, typically the total mass of each batch is fixed,each batch is a closed system, and the reaction or residence time forall reactants of the medium is the same. After discharging the batch,the fermentation chamber is cleaned and re-started with the medium 3304for another batch cycle. Separation or purification of the desiredproduct from the other constituents in the harvest broth 3312, mayinclude further processing, including refolding, altering affinity, ionexchange purification, alteration of hydrophobic interactions, gelfiltration chromatography, ultra filtration and/or diafiltration,depending on the target product.

For fed-batch fermentation, typically an initial, partial charge oraliquot of medium 3304 is added to the fermentation chamber, andsubsequently inoculated with cell culture 3304. The medium 3304 may beadded at measured rates during the remainder of the fermentation cycle.The cell mass and the broth 3312 are typically harvested only at the endof the cycle.

Following harvest and purification of the target product (step 3316),(typically once the cell culture 3310 has attained a peak cell growthdensity within the bioreactor 3306), the purified product 3318 (in somecases, a pharmaceutical drug or Active Pharmaceutical Ingredient, orAPI) is attained. The purified product may then be processed as desiredand optionally packaged in appropriate containers during a sterilepackaging process 3322 for transfer to a pharmaceutical manufacturingplant, or other facility. The purified product may then be used for anydesired purpose, including for prevention, treatment, and/or diagnosisof disease.

Plants and Animals as Reactors

In addition, a reactor may include a plant or animal, which is used togenerate a plant or animal product, or recombinant product. In certainembodiments, the plant or animal target product may be a naturallyoccurring product (e.g., food bearing crops or meat, or textile-relatedproducts such as cotton fibers, etc.), or the target product may be agenetically altered product (for example, therapeutic agents, such humangrowth hormone or insulin or other biologically active proteins andpolypeptides). A genetically altered or recombinant product may beproduced by a transgenic or genetically altered plant, animal, orcombination thereof.

Fish Culture

Fish (e.g., Tilapia fish) may be grown in aquaculture for food, or as atransgenic vehicle for production of a target product. The preferredtemperature range for optimum tilapia growth is 82°-86° F. Growthdiminishes significantly at temperatures below 68° F. and death willtypically occur below 50° F. Also, at temperatures below about 54° F.,the immune resistance of tilapia declines and the animals are easilysubjected to infection by bacteria, fungi, and parasites.

Twenty years ago, aquaculture researchers in Nigeria attempted tocorrelate dissolved oxygen concentrations in pond waiter with Tilapiagrowth rates. UN FAO reports: The study was conducted by examininggrowth rates of young Tialapia at high dissolved oxygen levels(approximately 7.0 ppm); at mid-level DO (approximately 3.5 ppm); and atlow DO levels (less than 2 ppm). The growth rates were determined bymeasuring the weight of the fish. The final increase in weight at theend of the research was 19 grams for the high DO level fish; 5 grams forthe mid-level DO fish; and 1.5 g for the low DO level fish. Thisrepresents to a 74% and 92% reduction in growth rates correlating to theDO levels. Thus, as the DO levels decrease, the feeding and waste outputalso decrease. It was observed that the Tilapia in the low DO levelwater break the surface of the water in order to access ambient oxygenrequired for survival.

The gas-enriched fluids of the present invention further includeoxygenated freshwater supplies in which the high dissolved oxygen levelsin the water are maintained for extended periods of time. According toparticular aspects, using the diffuser device of the present inventionin an aquaculture setting, dissolved oxygen levels of over 35 ppm can berecorded in 103° F. water without significantly stressing the aquaticlife.

Plant Growth

In addition to animal growth, the gas-enriched fluids of the presentinvention may be utilized for plant growth and development. Gases (suchas oxygen) are required for plant root respiration, which allows for therelease of energy for growth, as well as water and mineral uptake. Plantgrowth has been widely and unequivocally proven to be boosted bymaintaining high gas (e.g. oxygen and/or nitrogen) levels within theroot zone. In this regard, increasing gas delivery to plant root systemsrepresents a potential for crop improvement through boosting rootactivity. Likewise, in embodiments in which transgenic plants are grown,increasing gas delivery to the plants may provide for increasedproduction of the target product (such as a therapeutic orbiopharmaceutical product).

Hydroponic crops represent one exemplary system for production which maygreatly benefit from the gas-enrichment diffuser devices of the presentinvention through direct gas-enrichment (e.g. oxygenation) of thenutrient solution bathing the root zone. Hydroponic crops are typicallyproduced in a limited volume of growing media or root area and as suchneed constant replacement of gases (e.g. oxygen) within the root zone.Hydroponic crops such as lettuce, spinach, tomatoes, and cucumbers havealready demonstrated a direct and significant response to thegas-enriched nutrient solution. Some of these responses includeincreases in plant growth, increases in root volume, increases in plantyield, and higher quality produce. Thus, hydroponic systems may benefitfrom the gas-enriched fluids of the present invention.

Other hydroponic crops have had similar responses to gas-enrichment inthe root zone. However, at warm temperatures, crop production declinesdue to the increased requirement for gases (such as oxygen) in the rootzone. Thus, enrichment is effective for preventing gas-starvation ofroot cells, as well as boosting growth under less than favorable growingconditions.

Typically tropical crops that are able to be grown at high densities dueto high light levels and rapid rates of development (and high root zonetemperatures) have a gas requirement that is many times greater thanthose grown in more temperate climates. Thus, gas-enrichment will becomenecessary in many systems of horticulture production. Highly populatedcountries, which rely heavily on producing intensive horticultural cropsfor income and sustenance from very limited areas of land, will benefitgreatly from this technology.

Soil-based cropping systems can also benefit from the gas-enrichedsolutions of the present invention. Many crops are fed via drip,trickle, or furrow irrigation and could potentially benefit greatly fromthe use of gas-enriched irrigation water or fertigation solutions. Suchcrops include, but are not limited to: vegetables (tomatoes, salad cropssuch as lettuce, herbs, cucurbits), cut flowers, ornamental flowers,turf, vineyards, orchards, and long-term plantings. Gases, such asoxygen, can directly impact the health and growth of the plant but canalso act indirectly by increasing the bio-availability of gases (e.g.oxygen) at the root zone, and can also improve the health of the plantby promoting microbial life in the soil.

With regard to the microbial life in the soil, the microbial populationsare essential for mineral conversion in the soil and organic systems andoverall plant health through suppression of plant diseases. While thesemicrobes are beneficial and often essential for crop production, thepopulations also require gases (e.g. oxygen), which can compete with thegases for plant root cells. Thus, supplying gases (e.g. oxygen) to theplant roots in order to enable microbial life to flourish is vital toboth organically grown crops, as well as standard growing conditions.High rates of gases supplied to the growing media/soil in organicsystems would potentially speed up the rate of organic fertilizerconversion and mineralization of plant usable nutrients, thus increasingthe health and productivity of highly profitable organic crops.

In addition, the available land for growing crops represents a challengein many countries with limited resources or unsuitable soils.

In addition to hydroponic crops, the technology disclosed herein mayapply to seed germination, seed raising, cell transplant production,propagation from cuttings, sprout production, animal fodder production,soil based cropping, turf industries, ornamental plants, and medicinalplants.

Systems for Making Gas-Enriched Fluids

As shown here, exemplary oxygenation systems comprises a supply orreservoir of fluid which is drawn up and circulated through tubing orother conduits by a pump which subsequently delivers the fluid to themixer/diffuser. The mixer/diffuser may be of any number of variousembodiments including those set forth and described herein above. Thesediffusers significantly increase the amount of dissolved gas (e.g.oxygen) present in a fluid by introducing, for example, gaseous oxygento the fluid using a diffuser having coaxial cylindrical or frustoconical stator and rotor components rotating discs or plates within ahousing, Mazzie diffusers and impellers to create the desired cavitationand succussion desired for mixing of the fluid and the gas. It should benoted that many of the fluids will be aqueous or water-based, but thatthe present invention is not limited to these.

The diffuser is supplied with fluid by the pump and combines this with,for example, gaseous oxygen from supply and returns the oxygenated (orotherwise gas-enriched) fluid to the reservoir. The diffuser may employany number of possible embodiments for achieving diffusion including,but not limited to, micro-membrane, Mazzie injector, fine bubble,vortexing, electromolecular activation, or other methods. The oxygensupply may be either a cylinder of compressed oxygen gas or a system forgenerating oxygen gas from the air or other chemical components. Theoxygenated fluid produced by the diffuser is returned to the reservoirand may be recirculated through the pump and/or the diffuser again tofurther increase the dissolved oxygen content. Alternatively, the fluidmay be drawn off using the oxygenated fluid outlet. Oxygenated fluidswhich are drawn off through the outlet may be immediately put to use invarious applications or may be packaged for later use.

The packaging step may enclose gas-enriched (e.g. oxygenated) fluids ina variety of bottles, bags or other containers formed of plastic, metal,glass, or other suitable materials. Although the gas-enriched oroxygenated fluids produced in accordance with the present invention havea relatively long shelf life under atmospheric conditions, the shelflife may be further extended by using packaging which hermetically sealsthe gas-enriched fluid. In this manner, dissolved oxygen which works itsway out of the fluid during storage will form a pressure head above thegas-enriched fluid and help to prevent the migration of dissolvedoxygen, or other gas, out of the fluid and back into the atmosphere. Inone preferred embodiment of the present invention the gas-enriched fluidis packaged in an air tight container and the void space is filled withthe gas used for enrichment at a pressure of greater than one atmosphereprior to sealing the container. The packaging step may be used toproduce bottles, bags, pouches, or other suitable containers for holdingoxygenated solutions.

The presently disclosed systems and/or methods allow oxygen, or othergases, to be dissolved stably at a high concentration with minimalpassive loss. These systems and/or methods can be effectively used todissolve a wide variety of gases at heightened percentages into a widevariety of fluids. By way of example only, a deionized water at roomtemperature that typically has levels of about 7-9 ppm (parts permillion) of dissolved oxygen can achieve levels of dissolved oxygenranging from about 8-70 ppm using the disclosed systems and/or methods.In accordance with a particular exemplary embodiment, an oxygenatedwater or saline solution may be generated with levels of about 30-60 ppmof dissolved oxygen.

Culturing Chinese Hamster Ovary Cells

Chinese Hamster Ovary (CHO) cells are mammalian cells that arefrequently utilized in expression and production of recombinantproteins, particularly for those that require post-translationalmodification to express full biological function.

According to particular aspects, various characteristics of CHO cellscan be improved by integrating either a gas-enriching diffuser device100 or gas-enriched media produced by the device 100 and integrated intoa CHO bioreactor.

According to particular aspects, in the cultivation of CHO cells, it ispossible to utilize the gas-enriched fluids or media of the presentinvention including with a cell-line specific, serum-free medium (forexample from SAFC Biosciences, Inc.) for long-term growth of transformedCHO cells. According to additional aspects, CHO cells are not harmed bypassing through the gas-enrichment diffuser device in the process ofgas-enriching fluids (including media).

A test was conducted that measured the survival of CHO cells in aninline bioreactor. Briefly, the inline bioreactor was used with 2 L ofCHO media, and CHO cells at a density of 106 or higher. The bioreactorwas run for approximately 10 minutes (including the gas-enrichingdiffuser), and a 25 mL sample was removed. Cells were stained with 0.4%Trypan Blue, and cell viability was assessed with a hemacytometer.According to this measure, CHO cells were not significantly harmed bypassing through the gas-enrichment diffuser device in the process ofgas-enriching fluids (including media).

The foregoing described embodiments depict different componentscontained within, or connected with, different other components. It isto be understood that such depicted architectures are merely exemplary,and that in fact many other architectures can be implemented whichachieve the same functionality. In a conceptual sense, any arrangementof components to achieve the same functionality is effectively“associated” such that the desired functionality is achieved. Hence, anytwo components herein combined to achieve a particular functionality canbe seen as “associated with” each other such that the desiredfunctionality is achieved, irrespective of architectures or intermedialcomponents. Likewise, any two components so associated can also beviewed as being “operably connected,” or “operably coupled,” to eachother to achieve the desired functionality.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. Furthermore, it is to be understood that theinvention is solely defined by the appended claims. It will beunderstood by those within the art that, in general, terms used herein,and especially in the appended claims (e.g., bodies of the appendedclaims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to inventions containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”). The same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations).

Accordingly, the invention is not limited except as by the appendedclaims.

EXAMPLES Example 1 Dissolved Oxygen Stability

As indicated in FIG. 30, there is illustrated the dissolved oxygenlevels in a 500 ml thin-walled plastic bottle and a 1000 ml glass bottlewhich were each capped and stored at 65 degrees Fahrenheit.

As can be seen, when the plastic bottle is opened approximately 65 daysafter bottling, the dissolved oxygen level within the water isapproximately 27.5 ppm. When a second bottle is opened at approximately95 days after bottling, the dissolved oxygen level is approximately 25ppm. Likewise, for the glass bottle, the dissolved oxygen level isapproximately 40 ppm at 65 days and is approximately 41 ppm at 95 days.Thus, this chart indicates that the dissolved oxygen levels within bothplastic and glass bottles are maintained at relatively high rates at 650Fahrenheit when the oxygen is diffused within the fluid using thedescribed system and method.

Example 2 Decayed Oxygen Content in Balanced Salt Solution

FIG. 33 illustrates the dissolved oxygen retention of a 500 ml balancedsalt solution that originally had a dissolved oxygen level of 5 ppm.Following enrichment of the solution at standard temperature andpressure with the diffuser of the present invention, the dissolvedoxygen level was approximately 41 ppm. The solution was kept in an amberglass bottle. After an hour, the dissolved oxygen level was 40 ppm; 36ppm after two hours; 34 ppm after three hours; and slightly more than 30ppm after approximately four and a half hours. The final measurement wastaken shortly before six hours, at which point the dissolved oxygenlevel was approximately 28 ppm.

Example 3 Microbubble Size

Experiments were performed with a gas-enriched fluid by using thediffuser of the present invention in order to determine a gasmicrobubble size limit. The microbubble size limit was established bypassing the gas enriched fluid through 0.22 and 0.1 micron filters. Inperforming these tests, a volume of fluid passed through the diffuser ofthe present invention and generated a gas-enriched fluid. Sixtymilliliters of this fluid was drained into a 60 ml syringe. Thedissolved oxygen level of the fluid within the syringe was then measuredby Winkler titration. The fluid within the syringe was injected througha 0.22 micron Millipore Millex GP50 filter and into a 50 ml beaker. Thedissolved oxygen rate of the material in the 50 ml beaker was thenmeasured. The experiment was performed three times to achieve theresults illustrated in Table 5 below.

TABLE 5 DO levels DO AFTER 0.22 DO IN SYRINGE MICRON FILTER 42.1 ppm39.7 ppm 43.4 ppm 42.0 ppm 43.5 ppm 39.5 ppm

As can be seen, the dissolved oxygen levels that were measured withinthe syringe and the dissolved oxygen levels within the 50 ml beaker werenot significantly changed by passing the diffused material through a0.22 micron filter, which implies that the microbubbles of dissolved gaswithin the fluid are not larger than 0.22 microns.

A second test was performed in which a batch of saline solution wasenriched with the diffuser of the present invention and a sample of theoutput solution was collected in an unfiltered state. The dissolvedoxygen level of the unfiltered sample was 44.7 ppm. A 0.1 micron filterwas used to filter the oxygen-enriched solution from the diffuser of thepresent invention and two additional samples were taken. For the firstsample, the dissolved oxygen level was 43.4 ppm. For the second sample,the dissolved oxygen level was 41.4 ppm. Finally, the filter was removedand a final sample was taken from the unfiltered solution. In this case,the final sample had a dissolved oxygen level of 45.4 ppm. These resultswere consistent with those in which the Millipore 0.22 micron filter wasused. Thus, the majority of the gas bubbles or microbubbles within thesaline solution are approximately less than 0.1 microns in size.

Example 4 Sparging Effects

FIGS. 34 and 35 illustrate the sparging affects of the diffuser of thepresent invention on a fluid passing therethrough. The sparging ofoxygen-enriched water occurred in an 8 gallon tank at standardtemperature and pressure. As indicated, initially the oxygen-enrichedwater had a dissolved oxygen level of approximately 42 ppm. After 2minutes of running through the diffuser, the nitrogen had sparged theoxygen-enriched water such that the dissolved oxygen level was thenslightly more than 20 ppm. At 6 minutes, the dissolved oxygen level wasapproximately 6 ppm. The dissolved oxygen level of the oxygen-enrichedwater reached a minimum value slightly greater than zero (0) atapproximately 14 minutes after the beginning of the process. Thesefigures illustrate the manner in which nitrogen may be diffused intowater to sparge the oxygen from the water. However, any gas could beused within any fluid to sparge one gas from the other and diffuse theother gas into the fluid. The same experiment could utilize any hostfluid material, and any fluid infusion material.

Example 5 Rayleigh Effects

Fluids processed through the diffuser device described herein exhibitdifferences within the structure of the water when compared with normalunprocessed water. Gas-enriched water made by embodiments disclosedherein has been shown to have more Rayleigh scattering compared tounprocessed water.

In experiments conducted, samples of gas-enriched and non-enriched waterwere prepared and sent for optical analysis. The purpose of these testswas to determine whether there are any gross optical differences betweennormal (unprocessed) deionized water and water enriched by the diffuserdevice of the present invention.

The two samples, were coded to maintain their identities in secrecy, andonly after the tests were completed were the samples identified. The twosamples were placed in a laser beam of 633 nanometers according to thediagram illustrated in FIG. 37A. Sample B, which was gas-enriched fluidaccording to certain embodiments disclosed herein, exhibited scatteredlight regardless of its position relative to the laser source. TheSample B fluid had been sealed in glass bottles for approximately oneweek. After two to three hours of opening the bottle, the scatteringeffect disappeared. Thus, the structure of the gas-enriched fluid isoptically different from the structure of the unprocessed fluid. Theoptical effect is not directly related to dissolved oxygen levels sincethe dissolved oxygen level at the start was approximately 45 ppm and atthe end of the experiment was estimated to be approximately 32 ppm.Results are shown in FIG. 37B.

Example 6 Generation of Solvated Electrons

Additional evidence has also suggested that the enriching processgenerated by the diffuser device of the present invention results insolvated electrons within the gas-enriched fluid. Due to the results ofthe polarographic dissolved oxygen probes, it is believed that thediffused fluid exhibits an electron capture effect and thus the fluidmay include solvated electrons within the gas-enriched material.

There are two fundamental techniques for measuring dissolved oxygenlevels electrically: galvanic measuring techniques and polarographicmeasurements. Each process uses an electrode system wherein thedissolved oxygen levels within the solution being tested react with acathode of the probe to produce a current. Dissolved oxygen levelsensors consist of two electrodes, an anode and a cathode, which areboth immersed in electrolyte within the sensor body. An oxygen permeablemembrane separates the anode and cathode from the solution being tested.Oxygen diffuses across the membrane and interacts with the internalcomponents of the probe to produce an electrical current. The cathode isa hydrogen electrode and carries negative potential with respect to theanode. The electrolyte solution surrounds the electrode pair and iscontained by the membrane. When no oxygen is present, the cathode ispolarized by hydrogen and resists the flow of current. When oxygenpasses through the membrane, the cathode is depolarized and electronsare consumed. The cathode electrochemically reduces the oxygen tohydroxyl ions according to the following equation:

O₂+2H₂O+4E⁻=4OH⁻

When performing dissolved oxygen level measurements of a gas-enrichedsolution according to the systems of the present invention, an overflowcondition has been repeatedly experienced wherein the dissolved oxygenmeter displays a reading that is higher than the meter is capable ofreading. However, evaluation of the gas-enriched solution by WinklerTitration indicates lower dissolved oxygen (DO) level for the solutionthan indicated by the probe. Typically, a DO probe (such as the Orion862 used in these experiments) has a maximum reading of 60 ppm. However,when the meter is left in gas-enriched water of the present invention,it overflows.

Without wishing to be bound by any particular mechanism of action, themechanism of the meter responds to electrons where the oxygen reacts.However, according to electron spin resonance, no free ions are presentin the fluid. Thus, the fluid presumably contains solvated electronsstabilized by the oxygen species that is also present in the fluid.

Example 7 In Vitro Wound Healing

The effects of a gas-enriched fluid (enriched with oxygen) were testedfor the ability of cultured human epidermal keratinocytes to seal awound.

Human epidermal keratinocytes were isolated from neonatal foreskins thatwere obtained from routine circumcision and de-identified. Foreskinswere washed twice in PBS and incubated in 2.4 U/mL Dispase II in orderto separate the dermis from the epidermis. The epidermis was incubatedwith 0.25% trypsin/1 mM EDTA, neutralized with soy bean trypsininhibitor, agitated, and passed through a 70 um sieve to separate thecells. Next, the cell suspension was centrifuged and resuspended in cellculture medium (M154) supplemented with 0.07 mM CaCl₂, and humankeratinocyte growth supplements (0.2% hydrocortisone, 0.2 ng/mL humanepidermal growth factor) and penicillin/streptomycin, amphoteracinantibiotic cocktail. The keratinocyte cell suspensions were plated ontouncoated 12-well culture dishes and the medium replaced after 24 hours,and every 48 hours after the initial seeding.

Upon reaching cellular confluence, linear scratches were made with asterile p1000 pipette tip, which resulted in a uniform cell-free wound.The monolayers were washed several times with Dulbecco's PBS in order toremove any cellular debris. The wound monolayers were then incubated inthe following media: i) the complete growth media (as described above inthis Example); ii) the complete growth media diluted 1:1 with a shearedversion of saline without oxygen (control fluid that was processed usingthe disclosed diffuser device but without adding a gas); and iii) thecomplete growth media diluted 1:1 with oxygen-enriched saline. Eachstudy was done in triplicate.

Prior to incubation, the wells were filled with the respective media andsealed by placing a 25×25 mm glass coverslip on top of each well. At 6,12, 24, and 48 hours post-wounding, oxygen measurements were made, andcultures were imagined.

Six hours post-wounding, the edges of the wounds in the saline andgas-enriched media were more ruffled than those in the media controlthat was processed with the diffuser device disclosed herein, butwithout the addition of a gas. Twelve hours post-wounding the edges ofthe wounds in all three media appeared uneven, with keratinocytes alongthe borders migrating toward the center of the wounds. Quantification ofmigrating keratinocytes revealed approximately the same level ofkeratinocyte migration in the saline and gas-enriched media. Results ofthe experiment are shown in FIGS. 40A and 40B.

Example 8 Improved Wound Healing

A study was performed to determine the improved healing characteristicsof wounds that were exposed to an oxygen-enriched saline solution thatwas processed according to embodiments disclosed herein. In thisexperiment, bandages were placed on porcine dermal excision biopsywounds. The bandages soaked in oxygen-enriched saline solution or acontrol group of bandages soaked in a saline solution that was notoxygen-enriched. Microscopically, several factors were evaluated by thestudy including: 1) epidermalization; 2) neovascularization; 3)epidermal differentiation; 4) mast cell migration; and 5) mitosis.

Externally, the wounds appeared to heal at varying rates. The woundstreated with the oxygen-enriched saline solution showed an increase inwound healing at days 4 through 11. However, both wounds seemed tocomplete healing at approximately the same time. The study showed thatbetween days 3 and 11, the new epidermis in wounds treated with theoxygen-enriched saline solution migrated at two to four times as fast asthe epidermis of the wounds treated with the normal saline solution. Thestudy also showed that between 15 and 22 days, the wound treated by theoxygen-enriched saline solution differentiated at a more rapid rate asevidenced by the earlier formation of more mature epidermal layers. Atall stages, the thickening that occurs in the epidermis associated withnormal healing did not occur within the wounds treated by theoxygen-enriched saline solution.

Without wishing to be bound by any particular theory, it is believedthat the oxygen-enriched saline solution may increase the localizedlevel of NO within the wounds. NO modulates growth factors, collagendeposition, inflammation, mast cell migration, epidermal thickening, andneovascularization in wound healing. Furthermore, nitric oxide isproduced by an inducible enzyme that is regulated by oxygen.

Thus, while not wishing to be bound to any particular theory, theinventive gas-enriched fluid may stimulate NO production, which is inaccordance with the spectrum of wound healing effects seen in theseexperiments.

The epidermis of the healing pigs experienced earlier differentiation inthe oxygen-enriched saline group at days 15 through 22. In the case ofmast cell migration, differences also occurred in early and latemigration for the oxygen-enriched solution. A conclusive result for thelevel of mitosis was unascertainable due to the difficulty in staining.

Referring now to FIG. 41A through 41F, various illustrations compare thewound healing results of the porcine epidermal tissues with or withoutoxygen-enriched saline solution. Thus, the healing of the control woundand of the wound using the oxygen-enriched saline solution was followedfor days 1, 4 and 16. FIG. 41A illustrates the wound healing for thecontrol wound on day 1. As can be seen, the wound shows epidermal/dermalthickening and a loss of contour. FIG. 41B illustrates the wound healingon day 1 for the wound treated using the oxygen-enriched salinesolution. The wound shows normal epidermal/dermal thickness and normalcontouring is typical on a new wound.

Referring now to FIGS. 41C and 41D, there are illustrated the woundhealing for the control wound on day 4 and the wound healing for thewound treated with the oxygen-enriched saline solution on day 4. For thecontrol wound illustrated in FIG. 41C, the wound shows a 600 micronepidermal spur. In the wound treated with the oxygen-enriched salinesolution in FIG. 41D, there is illustrated a 1200 micron epidermal spur.Thus, in the first 4 days of the experiment, the epidermal spur createdin the wound treated using the oxygen-enriched saline solution shows anepidermal growth rate of twice of that of the wound that was not treatedwith the oxygen-enriched saline solution.

Referring now to FIG. 41E, there is illustrated the control wound at day16. The wound shows less differentiated epidermis with loss ofepidermal/dermal contour than that illustrated by the wound treated withthe oxygen-enriched saline solution illustrated in FIG. 41F. FIG. 41Fshows more differentiated epidermis and more normal epidermal/dermalcontouring in the wound.

Thus, as illustrated with respect to FIGS. 41A through 41F, the woundtreated with the oxygen-enriched saline solution shows much greaterhealing characteristics than the untreated wound and shows a greaterdifferentiated epidermis with more normal epidermal/dermal contour.

Example 9 Glutathione Peroxidase Study

The inventive oxygen-enriched fluid was tested for the presence ofhydrogen peroxide by testing the reactivity with glutathione peroxidaseusing a standard assay (Sigma). Water samples were tested by adding theenzyme cocktail and inverting. Continuous spectrophotometric ratedetermination was made at A₃₄₀ nm, and room temperature (25 degreesCelsius). Samples tested were: 1. deionized water (negative control), 2.inventive oxygen-enriched fluid at low concentration, 3. inventiveoxygen-enriched fluid at high concentration, 4. hydrogen peroxide(positive control).

The hydrogen peroxide positive control showed a strong reactivity, whilenone of the other fluids tested reacted with the glutathione peroxidase.

Example 10 Electrokinetically Generated Superoxygenated Fluids and Solaswere Shown to Provide for Synergistic Prolongation Effects (e.g.,Suppression of Bronchoconstriction) with Albuterol In Vivo in anArt-Recognized Animal Model of Human Bronchoconstriction (Human AsthmaModel)

Experiment 1:

In an initial experiment, sixteen guinea pigs were evaluated for theeffects of bronchodilators on airway function in conjunction withmethacholine-induced bronchoconstriction. Following determination ofoptimal dosing, each animal was dosed with 50 μg/mL to deliver thetarget dose of 12.5 μg of albuterol sulfate in 250 μL per animal.

The study was a randomized blocked design for weight and baseline PenHvalues. Two groups (A and B) received an intratracheal instillation of250 μL of 50 μg/mL albuterol sulfate in one or two diluents: Group A wasdeionized water that had passed through the inventive device, withoutthe addition of oxygen, while Group B was inventive gas-enriched water.Each group was dosed intratracheally with solutions using a Penn CenturyMicrosprayer. In addition, the animals were stratified across BUXCOplethysmograph units so that each treatment group is represented equallywithin nebulizers feeding the plethysmographs and the recording units.

Animals that displayed at least 75% of their baseline PenH value at 2hours following albuterol administration were not included in the dataanalyses. This exclusion criteria is based on past studies where thefailure to observe bronchoprotection with bronchodilators can beassociated with dosing errors. As a result, one animal from the controlgroup was dismissed from the data analyses.

Once an animal had greater than 50% bronchoconstriction, the animal wasconsidered to be not protected. As set forth in Table 6 below, 50% ofthe Group B animals (shaded) were protected from bronchoconstriction outto 10 hours (at which time the test was terminated).

TABLE 6 Bronchoconstriction Protection as Measured with MethacholineChallenge; Group A; Percent protection from bronchoconstriction byanimal number Time (hours) 1 2 3 4 5 6 7 0 100.00 100.00 100.00 100.00100.00 100.00 100.00 2 20.81 23.82 32.89 11.56 7.91 24.95 20.15 6 15.679.96 8.53 8.40 81.66 75.60 91.97 10 173.92 130.34 95.45 68.14 57.85103.95 69.03 Bronchoconstriction Protection as Measured withMethacholine Challenge; Group B; Percent protection frombronchoconstriction by animal number Time (hours) 1

3

7

0 100.00

100.00

100.00

2 15.85

17.88

21.33

6 211.57

68.79

118.26

10 174.54

88.16

123.47

Experiment 2: A Bronchoconstriction Evaluation of RDC1676 With AlbuterolSulfate in Male Hartley Guinea Pigs:

An additional set of experiments was conducted using a larger number ofanimals to evaluate the protective effects of the inventiveelectrokinetically generated fluids (e.g., RDC1676-00, RDC1676-01,RDC1676-02 and RDC1676-03) against methacholine-inducedbronchoconstriction when administered alone or as diluents for albuterolsulfate in male guinea pigs.

Materials:

Guinea Pigs (Cavia porcellus) were Hartley albino, Crl:(HA)BR fromCharles River Canada Inc. (St. Constant, Quebec, Canada). Weight:Approximately 325±50 g at the onset of treatment. Number of groups was32, with 7 male animals per group (plus 24 spares form same batch ofanimals). Diet; All animals had free access to a standard certifiedpelleted commercial laboratory diet (PMI Certified Guinea Pig 5026; PMINutrition International Inc.) except during designated procedures.

Methods:

Route of administration was intratracheal instillation via a PennCentury Microsprayer and methacholine challenge via whole bodyinhalation. The intratracheal route was selected to maximize lungexposure to the test article/control solution. Whole body inhalationchallenge has been selected for methacholine challenge in order toprovoke an upper airway hypersensitivity response (i.e.bronchoconstriction).

Duration of treatment was one day.

Table 7 shows the experimental design. All animals were subjected toinhalation exposure of methacholine (500 μg/ml), 2 hours followingTA/Control administration. All animals received a dose volume of 250 μl.Therefore, albuterol sulfate was diluted (in the control article and the4 test articles) to concentrations of 0, 25, 50 and 100 μg/ml.

Thirty minutes prior to dosing, solutions of albuterol sulfate of 4different concentrations (0, 25, 50 and 100 μg/ml) was made up in a 10×stock (500 μg/mL) in each of these four test article solutions(RDC1676-00, RDC1676-01, RDC1676-02; and RDC1676-03). Theseconcentrations of albuterol sulfate were also made up innon-electrokinetically generated control fluid (control 1). The dosingsolutions were prepared by making the appropriate dilution of each stocksolution. All stock and dosing solutions were maintained on ice onceprepared. The dosing was completed within one hour after thetest/control articles are made. A solution of methacholine (500 μg/ml)was prepared on the day of dosing.

Each animal received an intratracheal instillation of test or controlarticle using a Penn Century microsprayer. Animals were food deprivedovernight and were anesthetized using isoflurane, the larynx wasvisualized with the aid of a laryngoscope (or suitable alternative) andthe tip of the microsprayer was inserted into the trachea. A dose volumeof 250 μl/animal of test article or control was administered.

The methacholine aerosol was generated into the air inlet of a mixingchamber using aeroneb ultrasonic nebulizers supplied with air from aBuxco bias flow pump. This mixing chamber in turn fed four individualwhole body unrestrained plethysmographs, each operated under a slightnegative pressure maintained by means of a gate valve located in theexhaust line. A vacuum pump was used to exhaust the inhalation chamberat the required flow rate.

Prior to the commencement of the main phase of the study, 12 spareanimals were assigned to 3 groups (n=4/group) to determine the maximumexposure period at which animals may be exposed to methacholine toinduce a severe but non-fatal acute bronchoconstriction. Four animalswere exposed to methacholine (500 μg/mL) for 30 seconds and respiratoryparameters were measured for up to 10 minutes following commencement ofaerosol. Methacholine nebulizer concentration and/or exposure time ofaerosolization was adjusted appropriately to induce a severe butnon-fatal acute/reversible bronchoconstriction, as characterized by antransient increase in penes.

Once prior to test article administration (Day-1) and again at 2, 6, 10,14, 18, 22 and 26 hours postdose, animals were placed in the chamber andventilatory parameters (tidal volume, respiratory rate, derived minutevolume) and the enhanced pause Penh were measured for a period of 10minutes using the Buxco Electronics BioSystem XA system, followingcommencement of aerosol challenge to methacholine. Once animals werewithin chambers baseline, values were recorded for 1-minute, followingwhich methacholine, nebulizer concentration of 500 ug/mL wereaerosoloized for 30 seconds, animals were exposed to the aerosol forfurther 10 minutes during which time ventilatory paramaters werecontinuously assessed. Penh was used as the indicator ofbronchoconstriction; Penh is a derived value obtained from peakinspiratory flow, peak expiratory flow and time of expiration.Penh=(Peak expiratory flow/Peak inspiratory flow)*(Expiratory time/timeto expire 65% of expiratory volume−1).

Animals that did not display a severe acute broncoconstriction duringthe predose methacholine challenge were replaced. Any animal displayingat least 75% of their baseline PenhPenes value at 2 hours post dose werenot included in the data analysis. The respiratory parameters wererecorded as 20 second means.

Data considered unphysiological was excluded from further analysis.

Changes in Penh were plotted over a 15 minute period and Penh value wasexpressed as area under the curve. Numerical data was subjected tocalculation of group mean values and standard deviations (asapplicable).

TABLE 7 Experimental design; 7 male guinea pigs per group. AlbuterolAlbuterol Albuterol Albuterol (6/25 μg/ (12.5 μg/ (25 μg/ Group ID (0μg/animal) animal) animal) animal) 1 (control 1) 7 males 7 males 7 males7 males (ambient oxygen) 5 (RDC1676-00 7 males 7 males 7 males 7 males(Solas) 6 (RDC1676-01 7 males 7 males 7 males 7 males (20 ppm oxygen) 7(RDC1676-02 7 males 7 males 7 males 7 males (40 ppm oxygen) 8(RDC1676-03 7 males 7 males 7 males 7 males (60 ppm oxygen)

Results:

As shown in FIG. 107A-D, in the absence of Albuterol, administration ofthe inventive electrokinetically generated fluids had no apparent effecton mean percent baseline PenH values, when measured over a 26 hourperiod.

Surprisingly, however, as shown in FIG. 108A-D, administration ofalbuterol (representative data for the 25 μg albuterol/animal groups areshown) formulated in the inventive electrokinetically generated fluids(at all oxygen level values tested; ambient (FIG. 108-A), 20 ppm (FIG.108-B), 40 ppm (FIG. 108-C) and 60 ppm (FIG. 108-D)) resulted in astriking prolongation of anti-broncoconstrictive effects of albuterol,compared to control fluid. That is, the methacholine results showed aprolongation of the bronchodilation of albuterol out to at least 26hours. FIGS. 108 A-D shows that there were consistent differences at alloxygen levels between RDC1676 and the normal saline control. Combiningall 4 RDC1676 fluids, the p value for the overall treatment differencefrom normal saline was 0.03.

According to particular aspects of the present invention, therefore, theinventive electrokinetically generated solutions provide for synergisticprolongation effects with Albuterol, thus providing for a decrease in apatient's albuterol usage, enabling more efficient cost-effective druguse, fewer side effects, and increasing the period over which a patientmay be treated and responsive to treatment with albuterol.

Example 11 A Cytokine Profile was Determined

Mixed lymphocytes were obtained from a single healthy volunteer donor.Buffy coat samples were washed according to standard procedures toremove platelets. Lymphocytes were plated at a concentration of 2×10⁶per plate in RPMI media (+50 mm HEPES) diluted with either inventivegas-enriched fluid or distilled water (control). Cells were stimulatedwith 1 microgram/mL T3 antigen, or 1 microgram/mL phytohemagglutinin(PHA) lectin (pan-T cell activator), or unstimulated (negative control).Following 24-hour incubation, cells were checked for viability and thesupernatants were extracted and frozen.

The supernatants were thawed, centrifuged, and tested for cytokineexpression using a XMAP® (Luminex) bead lite protocol and platform.

Two million cells were plated into 6 wells of a 24-well plate in fullRPMI+50 mm Hepes with either inventive oxygen-enriched fluid (water)(wells 1, 3, and 5) or distilled water (2, 4 and 6) (10×RPMI dilutedinto water to make 1×). Cells were stimulated with 1 ug/ml T3 antigen(wells 1 and 2) or PHA (wells 3 and 4). Control wells 5 and 6 were notstimulated. After 24 hours, cells were checked for viability andsupernatants were collected and frozen. Next, the supernatants werethawed and spun at 8,000 g to pellet. The clarified supernatants wereassayed for the cytokines listed using a LUMINEX BEAD LITE™ protocol andplatform. The numerical data is tabulated in Table 8, and thecorresponding bar graphs are depicted in FIG. 38. Notably, IFN-γ levelwas higher in the inventive gas-enriched culture media with T3 antigenthan in the control culture media with T3 antigen, while IL-8 was lowerin the inventive gas-enriched culture media with T3 antigen than in thecontrol culture media with T3 antigen. Additionally, IL-6, IL-8, andTNF-α levels were lower in the inventive gas-enriched media with PHA,than in the control media with PHA, while IL-1β levels were lower in theinventive gas-enriched fluid with PHA when compared with control mediawith PHA. In the inventive gas-enriched media alone, IFN-γ levels werehigher than in control media.

TABLE 8 Sample IFN Il-10 Il-12p40 Il-12p70 Il-2 Il-4 Il-5 Il-6 Il-8Il-ib IP-10 TNFa 1 0 0 0 2.85 0 0 7.98 20.3 1350 7.56 11500 15.5 2 0 0 03.08 0 0 8 15.2 8940 3.68 4280 7.94 3 0 581 168 3.15 0 0 8 16400 22003280 862 13700 4 0 377 56.3 4.22 0 0 8.08 23800 22100 33600 558 16200 50 0 0 2.51 0 0 7.99 24 1330 7.33 5900 8.55 6 0 0 0 2.77 0 0 8 5.98 32104.68 3330 0

Example 12 Myelin Oligodendrocyte Glycoprotein (MOG)

As set forth in FIG. 48, lymphocyte proliferation in response to MOGantigenic peptide was increased when cultured in the presence of theinventive gas-enriched fluid when compared to pressurized, oxygenatedfluid (pressure pot) or deionized control fluid. Thus, the inventivegas-enriched fluid amplifies the lymphocyte proliferative response to anantigen to which the cells were previously primed.

Myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55)(M-E-V-G-W-Y-R-S-P-F-S-R-O-V-H-L-Y-R-N-G-K) (SEQ ID NO:1; seepublication US20080139674, incorporated by reference herein, includingfor purposes of this SEQ ID NO:1) corresponding to the known mousesequence was synthesized. Next, 5×10⁵ spleen cells were removed from MOGT cell receptor transgenic mice previously immunized with MOG, and werecultured in 0.2 ml TCM fluid reconstituted with inventive gas-enrichedfluid, pressurized oxygenated water (pressure pot water) or with controldeionized water. Splenocytes were cultured with MOG p35-55 for 48 or 72hours, respectively. Cultures were pulsed with 1 Ci [3H]-thymidine andharvested 16 hours later. Mean cpm of [3H] thymidine incorporation wascalculated for triplicate cultures. Results are shown in FIG. 48.

Example 13 Cytokine Expression

In particular aspects, human mixed lymphocytes were stimulated with T3antigen or PHA in inventive electrokinetic fluid, or control fluid, andchanges in IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40),IL-12(p70), IL-13, IL-17, Eotaxin, IFN-γ, GM-CSF, MIP-1β, MCP-1, G-CSF,FGFb, VEGF, TNF-α, RANTES, Leptin, TNF-β, TFG-β, and NGF were evaluated.As can be seen from FIG. 38, pro-inflammatory cytokines (IL-1β, TNF-α,IL-6, and GM-CSF), chemokines (IL-8, MIP-1α, RANTES, and Eotaxin),inflammatory enzymes (iNOS, COX-2, and MMP-9), allergen responses (MHCclass II, CD23, B7-1, and B7-2), and Th2 cytokines (IL-4, IL-13, andIL-5) tested were reduced in test fluid versus control fluid. Bycontrast, anti-inflammatory cytokines (e.g., IL1R-α, TIMPs) tested wereincreased in test fluid versus control fluid.

To expand on these data, Applicants used an art recognized model systeminvolving ovalbumin sensitization, for assessing allergichypersensitivity reactions. The end points studied were particularcytologic and cellular components of the reaction as well as serologicmeasurements of protein and LDH. Cytokine analysis was performed,including analysis of Eotaxin, IL-1A, IL-1B, KC, MCP-1, MCP-3, MIP-1A,RANTES, TNF-A, and VCAM.

Briefly, male Brown Norway rats were injected intraperitoneally with 0.5mL Ovalbumin (OVA) Grade V (A5503-1G, Sigma) in solution (2.0 mg/mL)containing aluminum hydroxide (Al (OH)₃) (200 mg/mL) once each on days1, 2, and 3. The study was a randomized 2×2 factorial arrangement oftreatments (4 groups). After a two week waiting period to allow for animmune reaction to occur, the rats were either exposed or were treatedfor a week with either RDC1676-00 (sterile saline processed through theRevalesio proprietary device), and RDC1676-01 (sterile saline processedthrough the Revalesio proprietary device with additional oxygen added).At the end of the 1 week of treatment for once a day, the 2 groups werebroken in half and 50% of the rats in each group received either Salineor OVA challenge by inhalation.

Specifically, fourteen days following the initial serialization, 12 ratswere exposed to RDC1676-00 by inhalation for 30 minutes each day for 7consecutive days. The air flow rate through the system was set at 10liters/minute. A total of 12 rats were aligned in the pie chamber, witha single port for nebulized material to enter and evenly distribute tothe 12 sub-chambers of the Aeroneb.

Fifteen days following initial sensitization, 12 rats were exposed toRDC 1676-01 by ultrasonic nebulization for 30 minutes each day for 7consecutive days. The air flow was also set for 10 liters/minute, usingthe same nebulizer and chamber. The RDC 1676-00 was nebulized first andthe Aeroneb chamber thoroughly dried before RDC 1676-01 was nebulized.

Approximately 2 hours after the last nebulization treatment, 6 rats fromthe RDC 1676-00 group were re-challenged with OVA (1% in saline)delivered by intratreacheal instillation using a Penn CenturyMicrosprayer (Model 1A-1B). The other 6 rats from the RDC 1676-00 groupwere challenged with saline as the control group delivered by way ofintratreacheal instillation. The following day, the procedure wasrepeated with the RDC 1676-01 group.

Twenty four hours after re-challenge, all rats in each group wereeuthanized by overdose with sodium pentobarbital. Whole blood sampleswere collected from the inferior vena-cava and placed into two disparateblood collection tubes: Qiagen PAXgene™ Blood RNA Tube and QiagenPAXgene™ Blood DNA Tube. Lung organs were processed to obtainbronchoalveolar lavage (BAL) fluid and lung tissue for RT-PCR to assesschanges in markers of cytokine expression known to be associated withlung inflammation in this model. A unilateral lavage technique was beemployed in order to preserve the integrity of the 4 lobes on the rightside of the lung. The left “large” lobe was lavaged, while the 4 rightlobes were tied off and immediately placedinot TRI-zol™, homogenized,and sent to the lab for further processing.

BAL analysis. Lung lavage was collected and centrifuged for 10 minutesat 4° C. at 600-800 g to pellet the cells. The supernatants weretransferred to fresh tubes and frozen at −80° C. Bronchial lavage fluid(“BAL”) was separated into two aliquots. The first aliquot was spundown, and the supernatant was snap frozen on crushed dry ice, placed in−80° C., and shipped to the laboratory for further processing. Theamount of protein and LDH present indicates the level of blood serumprotein (the protein is a serum component that leaks through themembranes when it's challenged as in this experiment) and cell death,respectively. The proprietary test side showed slight less protein thanthe control.

The second aliquot of bronchial lavage fluid was evaluated for totalprotein and LDH content, as well as subjected to cytologicalexamination. The treated group showed total cells to be greater than thesaline control group. Further, there was an increase in eosinophils inthe treated group versus the control group. There were also slightlydifferent polymorphonuclear cells for the treated versus the controlside.

Blood analysis. Whole blood was analyzed by transfer of 1.2-2.0 mL bloodinto a tube, and allowing it to clot for at least 30 minutes. Theremaining blood sample (approximately 3.5-5.0 mL) was saved for RNAextraction using TRI-zol™ or PAXgene™. Next, the clotted blood samplewas centrifuged for 10 minutes at 1200 g at room temperature. The serum(supernatant) was removed and placed into two fresh tubes, and the serumwas stored at −80° C.

For RNA extraction utilizing Tri-Reagent (TB-126, Molecular ResearchCenter, Inc.), 0.2 mL of whole blood or plasma was added to 0.75 mL ofTRI Reagent BD supplemented with 20 μL of 5N acetic acid per 0.2 mL ofwhole blood or plasma. Tubes were shaken and stored at −80° C. UtilizingPAXgene™, tubes were incubated for approximately two hours at roomtemperature. Tubes were then placed on their side and stored in the −20°C. freezer for 24 hours, and then transferred to −80° C. for long termstorage.

Luminex analysis. By Luminex platform, a microbead analysis was utilizedas a substrate for an antibody-related binding reaction which is readout in luminosity units and can be compared with quantified standards.Each blood sample was run as 2 samples concurrently. The units ofmeasurement are luminosity units and the groups are divided up into OVAchallenged controls, OVA challenged treatment, and saline challengedtreatment with proprietary fluid.

For Agilant gene array data generation, lung tissue was isolated andsubmerged in TRI Reagent (TR118, Molecular Research Center, Inc.).Briefly, approximately 1 mL of TRI Reagent was added to 50-100 mg oftissue in each tube. The samples were homogenized in TRI Reagent, usingglass-Teflon™ or Polytron™ homogenizer. Samples were stored at −80° C.

Blood Samples:

FIGS. 49-58 show the results of whole blood sample evaluations.

Exemplary FIG. 49 shows the basic luminosity data presentation formatfor the blood sample data. Letters designating the identity of themeasured cytokine (in this case KC) are at the top right of each datafigure. The data is presented both as data points (upper graph) and bargraphs (lower graph) of the individual samples. In either case, thegraphs are divided, from left to right, in four groups. The first 2groups (RDC1676-00 OVA and RDC1676-01 OVA, respectively) were those thatwere re-challenged with OVA by inhalation, whereas the last two groups(RDC1676-00 OVA and RDC1676-01 OVA, respectively) where those that werere-challenged with saline control only. Again, the suffix 00 representssaline treatment and suffix 01 represents inventive electrokinetic fluidtreated groups.

Each blood sample was split into 2 samples and the samples were runconcurrently. The units of measure are units of luminosity and thegroups, going from left to right are: OVA challenged controls; OVAchallenged inventive electrokinetic fluid treatment; followed by salinechallenged saline treatment; and saline challenged inventiveelectrokinetic fluid treatment. To facilitate review, both theRDC1676-01 groups are highlighted with gray shaded backdrops, whereasthe control saline treatment groups have unshaded backdrops.

Generally, in comparing the two left groups, while the spread of theRDC1676-01 group data is somewhat greater, particular cytokine levels inthe RDC1676-01 group as a whole are less than the samples in the controltreated group; typically about a 30% numerical difference between the 2groups. Generally, in comparing the right-most two groups, theRDC1676-01 group has a slightly higher numerical number compared to theRDC1676-00 group.

FIG. 50 shows analysis of RANTES (IL-8 super family) in blood sampledata according to particular exemplary aspects. Luminosity units for theleftmost two groups (the OVA challenged groups) indicate that generallyvalues in the RDC1676-01 treated group were less than the RDC1676-00control group as shown by the dot plot in the upper graph portion whichagain shows a 30-35% differential between the two groups, whereas in thesaline only exposed groups the cytokine level values where roughly thesame, or perhaps slightly increased in the RDC1676-01 treated group.

FIG. 51 shows analysis of MCP-1 in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 52 shows analysis of TNF alpha in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 53 shows analysis of MIP-1 alpha in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 54 shows analysis of IL-1 alpha in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 55 shows analysis of Vcam in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 56 shows analysis of IL-1 beta in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIGS. 57 and 58 show analysis of Eotaxin and MCP-3, respectively, inblood sample data according to particular exemplary aspects. In eachcase, luminosity units for the leftmost two groups (the OVA challengedgroups) indicate that generally values in the RDC1676-01 treated groupwere less than the RDC1676-00 control group as shown by the dot plot inthe upper graph portion, whereas in the saline only exposed groups thecytokine level values where roughly the same, or perhaps slightlyincreased in the RDC1676-01 treated group.

Bronchial Lavage Samples:

FIGS. 59-68 show the corresponding results of bronchoalveolar lavagefluid (BAL) sample evaluations.

FIG. 59 shows analysis of KC in BAL data according to particularexemplary aspects. In this instance the response level, coupled withsampling variability, was inconclusive with respect to a differencebetween the RDC1676-01 and RDC1676-00-treated groups; that is, KC showedrelatively little difference between the 2 groups, but the units ofluminosity were very small.

Likewise, FIG. 60 shows analysis of RANTES in BAL data according toparticular exemplary aspects, and showing marked variability in theRDC1676-01 group with one reading being markedly higher than the others,skewing the results.

Likewise, FIG. 61 shows analysis of TNF alpha in BAL data according toparticular exemplary aspects, and showing relatively little significancein the way of difference between the RDC1676-01 and RDC1676-00-treatedgroups.

FIG. 62 shows analysis of MCP-1 in BAL data according to particularexemplary aspects, and showing relatively little significance in the wayof difference between the RDC1676-01 and RDC1676-00-treated groups.

FIGS. 63 through 68 show analysis of MIP1-A, IL-1 alpha, Vcam, IL-1beta, MCP-3, and Eotaxin, respectively, in BAL data according toparticular exemplary aspects, and showing relatively little significancein the way of difference between the RDC1676-01 and RDC1676-00-treatedgroups.

In summary, this standard assay of inflammatory reaction to a knownsensitization produced, at least in the blood samples, a marked clinicaland serologic affect. Additionally, while significant numbers of controlanimals were physiologically stressed and nearly dying in the process,none of the RDC1676-01 treated group showed such clinical stresseffects. This was reflected then in the circulating levels of cytokines,with approximately 30% differences between the RDC1676-01-treated andthe RDC1676-01-treated groups in the OVA challenged groups. By contrast,there were small and fairly insignificant changes in cytokine, cellularand serologic profiles between the RDC1676-01-treated and theRDC1676-01-treated groups in the non-OVA challenged groups, which likelymerely represent minimal baseline changes of the fluid itself.

Example 14 Bradykinin B2 Receptor Affinity Binding

A Bio-Layer Interferometry biosensor, Octet Rapid Extended Detection(RED) (forteBio™) was utilized in order to examine membrane receptoraffinity binding of Bradykinin ligand with the Bradykinin B2 receptor.The biosensor system consists of a polished fiber optic embedded into apolypropylene hub with a sensor-specific chemistry at the tip. Thebiosensor set-up has a layer of molecules attached to the tip of anoptic fiber that creates an interference pattern at the detector. Anychange in the number of molecules bound causes a measured shift in thepattern of light.

As shown in FIG. 69 the Bradykinin B2 membrane receptor was immobilizedonto aminopropylsilane (APS) biosensor. The sample plate set up wasdesignated in FIG. 69 and analyzed in FIG. 70. Next, the binding ofBradykinin to the immobilized receptor was assessed according to thesample set up as designated in FIG. 71. Results of Bradykinin bindingare shown in FIG. 72. Bradykinin binding to the receptor was furthertitrated according to the set-up as designated in FIG. 73.

As indicated in FIG. 74, Bradykinin binding to the B2 receptor wasconcentration dependent, and binding affinity was increased in theproprietary gas-enriched saline fluid of the instant disclosure comparedto normal saline. Stabilization of Bradykinin binding to the B2 receptoris shown in FIG. 75.

Example 15 A Regulatory T-Cell Assay was Used to Show Effects of theInventive Electrokinetically Generated Fluids in Modulation of T-CellProliferation and Elaboration of Cytokines (II-10) and Other Proteins(e.g., GITR, Granzyme A, XCL1, pStat5, and Foxp3)) in Regulatory T-CellAssays, and of, for Example, Tryptase in PBMC

The ability of particular embodiments disclosed herein to regulate Tcells was studied by irradiating antigen presenting cells, andintroducing antigen and T cells. Typically, these stimulated T cellsproliferate. However, upon the introduction of regulatory T cells, theusual T cell proliferation is suppressed.

Methods:

Briefly, FITC-conjugated anti-CD25 (ACT-1) antibody used in sorting waspurchased from DakoCytomation (Chicago, Ill.). The other antibodies usedwere as follows: CD3 (HIT3a for soluble conditions), GITR (PEconjugated), CD4 (Cy-5 and FITC-conjugated), CD25 (APC-conjugated), CD28(CD28.2 clone), CD127-APC, Granzyme A (PE-conjugated), FoxP3(BioLegend), Mouse IgG1 (isotype control), and XCL1 antibodies. Allantibodies were used according to manufacturer's instructions.

CD4+ T cells were isolated from peripheral whole blood with CD4+ RosetteKit (Stemcell Technologies). CD4+ T cells were incubated withanti-CD127-APC, anti-CD25-PE and anti-CD4-FITC antibodies. Cells weresorted by flow cytometry using a FACS Aria into CD4+ CD25hiCD127lo/nTregand CD4+ CD25-responder T cells.

Suppression assays were performed in round-bottom 96 well microtiterplates. 3.75×103 CD4+ CD25neg responder T cells, 3.75×103 autologous Treg, 3.75×104 allogeneic irradiated CD3-depleted PBMC were added asindicated. All wells were supplemented with anti-CD3 (clone HIT3a at 5.0ug/ml). T cells were cultured for 7 days at 37° C. in RPMI 1640 mediumsupplemented with 10% fetal bovine serum. Sixteen hours before the endof the incubation, 1.0 mCi of ³H-thymidine was added to each well.Plates were harvested using a Tomtec cell harvester and ³H-thymidineincorporation determined using a Perkin Elmer scintillation counter.Antigen-presenting cells (APC) consisted of peripheral blood mononuclearcells (PBMC) depleted of T cells using StemSep human CD3+ T celldepletion (StemCell Technologies) followed by 40 Gy of irradiation.

Regulatory T cells were stimulated with anti-CD3 and anti-CD28conditions and then stained with Live/Dead Red viability dye(Invitrogen), and surface markers CD4, CD25, and CD127. Cells were fixedin the Lyze/Fix PhosFlow™ buffer and permeabilized in denaturingPermbuffer III®. Cells were then stained with antibodies against eachparticular selected molecule.

Statistical analysis was performed using the GraphPad Prism software.Comparisons between two groups were made by using the two-tailed,unpaired Student's t-test. Comparisons between three groups were made byusing 1-way ANOVA. P values less than 0.05 were considered significant(two-tailed). Correlation between two groups were determined to bestatistically significant via the Spearman coefficient if the r valuewas greater than 0.7 or less than −0.7 (two-tailed).

Results:

As indicated in FIG. 76, regulatory T cell proliferation was studied bystimulating cells with diesel exhaust particulate matter (PM, from EPA).The x-axis of FIG. 76 shows activated autologous CD4+ effector T cells(responder cells) as a solid black bar, and regulatory T cells alone inthe gray bar (shown for confirmation of anergy) which were mixed at a1:1 ratio as shown in the white bar. The y axis shows proliferation asmeasured by uptake of ³H-thymidine. As shown from left to right alongthe x-axis, “PM” indicates diesel exhaust derived Particulate Matter,“PM+Rev” indicates PM plus a gas-enriched electrokinetically generatedfluid (Rev) of the instant disclosure, “Solas” indicates anelectrokinetically generated fluid of the instant disclosure and devicethat is not gas-enriched beyond ambient atmosphere, only (no PM added),“Rev” indicates Rev alone (no PM added) as defined above, “Media”indicates the cell growth media alone control (minus PM; no Rev, noSolas), and “Saline Con” indicates the saline control (minus PM; no Rev,no Solas), “V” indicates verapamil, and “P” indicates propanolol, and“DT” is DT390 at 1:50.

As shown in FIG. 77, cells stimulated with PM (no Rev, no Solas)resulted in a decrease in secreted IL-10, while cells exposed to PM inthe presence of the fluids of the instant disclosure (“PM+Rev”) resultedin a maintained or only slightly decreased production of IL-10 relativeto the Saline and Media controls (no PM). Furthermore, Diphtheria toxin(DT390, a truncated diphtheria toxin molecule; 1:50 dilution of std.commercial concentration) was titrated into inventive fluid samples, andblocked the Rev-mediated effect of increase in IL-10 in FIG. 77. Notethat treatment with Rev alone resulted in higher IL-10 levels relativeto Saline and Media controls.

Likewise, similar results, shown in FIGS. 78-82, were obtained withGITR, Granzyme A, XCL1, pStat5, and Foxp3, respectively. In Figures,“NSC” is the same as “Solas” (no PM).

FIG. 83 shows AA PBMC data, obtained from an allergic asthma (AA)profile of peripheral blood mononuclear cells (PBMC) evaluatingtryptase. The AA PBMC data was consistent with the above T-regulatorycell data, as cells stimulated with particulate matter (PM) showed highlevels of tryptase, while cells treated with PM in the presence of thefluids of the instant disclosure (“PM+Rev”) resulted in significantlylower tryptase levels similar to those of the Saline and Media controls.Consistent with the data from T-regulatory cells, exposure to DT390blocked the Rev-mediated effect on tryptase levels, resulting in anelevated level of tryptase in the cells as was seen for PM alone (minusRev, no Rev, no Solas). Note that treatment with Rev alone resulted inlower tryptase levels relative to Saline and Media controls.

In summary, the data of FIG. 76, showing a decreased proliferation inthe presence of PM and Rev relative to PM in control fluid (no Rev, noSolas), indicates that the inventive electrokinetically generated fluidRev improved regulatory T-cell function as shown by relatively decreasedproliferation in the assay. Moreover, the evidence of this Example andFIGS. 76-83, indicate that beta blockade, GPCR blockade and Ca channelblockade affects the activity of Revera on Treg function.

Example 16 Treatment of Primary Bronchial Epithelial Cells (BEC) withthe Inventive Electrokinetically Generated Fluids Resulted in ReducedExpression and/or Activity of Two Key Proteins of the AirwayInflammatory Pathways, MMP9 and TSLP

Overview. As shown in Example 14 above (e.g., FIG. 75, showingStabilization of Bradykinin binding to the B2 receptor using Bio-LayerInterferometry biosensor, Octet Rapid Extended Detection (RED)(forteBio™)), Bradykinin binding to the B2 receptor was concentrationdependent, and binding affinity was increased in the electrokineticallygenerated fluid (e.g., Rev; gas-enriched electrokinetically generatedfluid) of the instant disclosure compared to normal saline.Additionally, as shown in Example 15 in the context of T-regulatorycells stimulated with diesel exhaust particulate matter (PM, standardcommercial source), the data showed a decreased proliferation ofT-regulatory cells in the presence of PM and Rev relative to PM incontrol fluid (no Rev, no Solas) (FIG. 76), indicating that theinventive electrokinetically generated fluid Rev improved regulatoryT-cell function; e.g., as shown by relatively decreased proliferation inthe assay. Moreover, exposure to the inventive fluids resulted in amaintained or only slightly decreased production of IL-10 relative tothe Saline and Media controls (no PM). Likewise, in the context of theallergic asthma (AA) profiles of peripheral blood mononuclear cells(PBMC) stimulated with particulate matter (PM), the data showed thatexposure to the fluids of the instant disclosure (“PM+Rev”) resulted insignificantly lower tryptase levels similar to those of the Saline andMedia controls. Additionally, the Diphtheria toxin (DT390, a truncateddiphtheria toxin molecule; 1:50 dilution of std. commercialconcentration) effects shown in Example 15 and FIGS. 76-83, indicatethat beta blockade, GPCR blockade and Ca channel blockade affects theactivity of the electrokinetically generated fluids on Treg and PBMCfunction. Furthermore, the data of Example 18 shows that, according toadditional aspects, upon exposure to the inventive fluids, tightjunction related proteins were upregulated in lung tissue. FIGS. 85-89show upregulation of the junction adhesion molecules JAM 2 and 3, GJA1,3, 4 and 5 (junctional adherins), OCLN (occludin), claudins (e.g., CLDN3, 5, 7, 8, 9, 10), TJP1 (tight junction protein 1), respectively.Furthermore, as shown in the patch clamp studies of Example 23, theinventive electrokinetically generated fluids (e.g., RNS-60) affectmodulation of whole cell conductance (e.g., under hyperpolarizingconditions) in Bronchial Epithelial Cells (BEC; e.g., Calu-3), andaccording to additional aspects, modulation of whole cell conductancereflects modulation of ion channels.

In this Example, Applicants have extended these discoveries byconducting additional experiments to measure the effects of productionof two key proteins of the airway inflammatory pathways. Specifically,MMP9 and TSLP were assayed in primary bronchial epithelial cells (BEC).

Materials and Methods:

Commercially available primary human bronchial epithelial cells (BEC)(HBEpC-c from Promocell, Germany) were used for these studies.Approximately 50,000 cells were plated in each well of a 12 well plateuntil they reached ˜80% confluence. The cells were then treated for 6hours with normal saline, control fluid Solas or the test fluid Revera60 at a 1:10 dilution (100 ul in 1 ml of airway epithelial growthmedium) along with the diesel exhaust particulate matter (DEP or PM)before being lifted for FACS analysis, as described in Example 8 herein.Both MMP9 and TSLP receptor antibodies were obtained from BD Biosciencesand used as per manufacturer's specifications.

Results:

In FIGS. 115 and 116, DEP represents cells exposed to diesel exhaustparticulate matter (PM, standard commercial source) alone, “NS”represents cells exposed to normal saline alone, “DEP+NS” representcells treated with particulate matter in the presence of normal saline,“Revera 60” refers to cells exposed only to the test material,“DEP+Revera 60” refer to cells treated with particulate matter in thepresence of the test material Revera 60. In addition, “Solas” and“DEP+Solas” represents cells exposed to the control fluid Solas alone orin combination with the particulate matter, respectively.

FIG. 115 shows that the test material Revera 60 reduces DEP induced TSLPreceptor expression in bronchial epithelial cells (BEC) by approximately90%. Solas resulted in a 55% reduction in TSLP receptor expression,while Normal saline failed to produce similar level of reduction in TSLPreceptor expression (approximately 20% reduction). The effect of theinventive solution in reducing TSLP receptor expression is a significantdiscovery in view of recent findings showing that TSLP plays a pivotalrole in the pathobiology of allergic asthma and local antibody mediatedblockade of TSLP receptor function alleviated allergic disease (Liu, YJ, Thymic stromal lymphopoietin: Master switch for allergicinflammation, J Exp Med 203:269-273, 2006; Al-Shami et al., A role forTSLP in the development of inflammation in an asthma model, J Exp Med202:829-839, 2005; and Shi et al., Local blockade of TSLP receptoralleviated allergic disease by regulating airway dendritic cells, ClinImmunol. 2008, Aug. 29. (Epub ahead of print)).

Likewise, FIG. 116 shows the effect of Revera 60, Solas and normalsaline on the DEP-mediated increase in MMP 9. Specifically, Revera 60inhibited the DEP-induced cell surface bound MMP9 levels in bronchialepithelial cells by approximately 80%, and Solas had an inhibitoryeffect of approximately 70%, whereas normal saline (NS) had a marginaleffect of about 20% reduction. MMP-9 is one of the major proteinasesinvolved in airway inflammation and bronchial remodeling in asthma.Recently, it has been demonstrated that the levels of MMP-9 aresignificantly increased in patients with stable asthma and even higherin acute asthmatic patients compared with healthy control subjects.MMP-9 plays a crucial role in the infiltration of airway inflammatorycells and the induction of airway hyperresponsiveness indicating thatMMP-9 may have an important role in inducing and maintaining asthma(Vignola et al., Sputum metalloproteinase-9/tissue inhibitor ofmetalloproteinase-1 ratio correlates with airflow obstruction in asthmaand chronic bronchitis, Am J Respir Crit. Care Med 158:1945-1950, 1998;Hoshino et al., Inhaled corticosteroids decrease subepithelial collagendeposition by modulation of the balance between matrixmetalloproteinase-9 and tissue inhibitor of metalloproteinase-1expression in asthma, J Allergy Clin Immunol 104:356-363, 1999; Simpsonet al., Differential proteolytic enzyme activity in eosinophilic andneutrophilic asthma, Am J Respir Crit Care Med 172:559-565, 2005; Lee etal., A murine model of toluene diisocyanate-induced asthma can betreated with matrix metalloproteinase inhibitor, J Allergy Clin Immunol108:1021-1026, 2001; and Lee et al., Matrix metalloproteinase inhibitorregulates inflammatory cell migration by reducing ICAM-1 and VCAM-1expression in a murine model of toluene diisocyanate-induced asthma, JAllergy Clin Immunol 2003; 111:1278-1284).

According to additional aspects, therefore, the inventiveelectrokinetically generated fluids have substantial therapeutic utilityfor modulating (e.g., reducing) TSLP receptor expression and/or forinhibiting expression and/or activity of MMP-9, including, for example,for treatment of inflammation and asthma.

Example 17 The Inventive Electrokinetically Generated Fluids were Shownto have a Synergistic Anti-Inflammatory Effect with Budesonide in anArt-Recognized Animal Model for Allergic Asthma

This working Example describes experiments performed to assess theairway anti-inflammatory properties of the inventive electrokineticallygenerated fluids (e.g., RDC-1676-03) in a Brown Norway rat ovalbuminsensitization model. The Brown Norway rat is an art-recognized model fordetermining the effects of a test material on airway function and thisstrain has been widely used, for example, as a model of allergic asthma.Airway pathology and biochemical changes induced by ovalbuminsensitization in this model resemble those observed in man (Elwood etal., J Allergy Clin Immuno 88:951-60, 1991; Sirois & Bissonnette, ClinExp Immunol 126:9-15, 2001). The inhaled route was selected to maximizelung exposure to the test material or the control solution. Theovalbumin-sensitized animals were treated with budesonide alone or incombination with the test material RDC 1676-03 for 7 days prior toovalbumin challenge. 6 and 24 hours following the challenge, total bloodcount and levels of several pro and anti-inflammatory cytokines as wellas various respiratory parameters were measured to estimate anybeneficial effect of administering the test material on variousinflammatory parameters.

Materials and Methods:

Brown Norway rats of strain Bn/Crl were obtained from Charles RiverKingston, weighing approximately 275±50 g at the onset of theexperiment. All animal studies were conducted with the approval byPCS-MTL Institutional Animal Care and Use Committee. During the study,the use and care of animals were conducted according to guidelines ofthe USA National Research Council as well as Canadian Council of AnimalCare.

Sensitization. On day 1 of the experiment, animals (14 animals in eachtreatment group) were sensitized by administration of a 1 mlintraperitoneal injection of a freshly prepared solution of 2 mgovalbumin/100 mg Aluminum Hydroxide per 1 ml of 0.9% Sodium Chloride,followed by repeat injection on day 3.

Treatment. Fifteen days following the initial sensitization, animalswere subjected to nebulized exposure to control (Normal saline) or testsolutions (electrokinetically generated fluids RDC1676-00, RDC1676-02and RDC-1676-03), either administered alone or in combination withBudesonide, once daily for 15 minutes for 7 consecutive days. Animalswere dosed in a whole body chamber of approximately 20 L, and testatmosphere was generated into the chamber air inlet using aeronebultrasonic nebulizers supplied with air from a Buxco bias flow pump. Theairflow rate was set at 10 liters/min.

Ovalbumin challenge. On day 21, 2 hours following treatment with thetest solutions, all animals were challenged with 1% ovalbumin nebulizedsolution for 15 minutes (in a whole body chamber at airflow 2 L/min).

Sample collection. At time points of 6 and 24 hours after the ovalbuminchallenge, blood samples were collected for total and differential bloodcell counts as well as for measuring levels of various pro andanti-inflammatory cytokines. In addition, Immediately after and at 6 and24 hours following ovalbumin challenge the enhanced pause Penh and tidalvolume were measured for a period of 10 minutes using the BuxcoElectronics BioSystem XA system.

Results:

Eosinophil Count: As expected, and shown in FIG. 109, treatment withBudesonide (“NS+Budesonide 750 μg/Kg”; densely crosshatched bar graph)reduced the total eosinophil count in the challenged animals relative totreatment with the normal saline “NS” alone control (open bar graph).Additionally, while treatment with the inventive fluid “RDC1676-03”alone (lightly crosshatched bar graph) did not significantly reduce theeosinophil count, it nonetheless displayed a substantial synergy withBudesonide in reducing the eosinophil count (“RDC1676-03+Budesonide 750μg/Kg”, solid dark bar graph). Similarly, in FIG. 110, the Eosinophil %also reflected a similar trend. While RDC1676-03 (lightly crosshatchedgraph bar) or Budesonide 750 ug/kg (densely crosshatched bar graph)alone did not have a significant effect on Eosinophil % count in thechallenged animals, the two in combination reduced the Eosinophil %significantly (solid dark bar graph).

Therefore, FIGS. 109 and 110 show, according to particular aspects ofthe present invention that the inventive electrokinetically generatedfluids (e.g., RDC1676-03) were demonstrated to have a substantialsynergistic utility in combination with Budesonide to significantlyreduce eosinophil count (“Eosinophil %” and total count) in anart-recognized rat model for human allergic asthma.

Respiratory Parameters:

FIGS. 111A-C and 112 A-C demonstrate the observed effect of the testfluids on Penh and tidal volume as measured immediately, 6 and 24 hoursafter the ovalbumin challenge. Penh is a derived value obtained frompeak inspiratory flow, peak expiratory flow and time of expiration andlowering of penh value reflects a favorable outcome for lung function.

Penh=(Peak expiratory flow/Peak inspiratory flow)*(Expiratory time/timeto expire 65% of expiratory volume−1).

As evident from FIGS. 111A-C, treatment with Budesonide (at both 500 and750 ug/kg) alone or in combination with any of the test fluids failed tosignificantly affect the Penh values immediately after the challenge.However, 6 hours after the challenge, animals treated with RDC1676-03alone or in combination with Budesonide 500 or 750 ug/kg demonstrated asignificant drop in Penh values. Although the extent of this drop wasdiminished by 24 hours post challenge, the trend of a synergistic effectof Budesonide and RDC fluid was still observed at this time point.

Tidal volume is the volume of air drawn into the lungs duringinspiration from the end-expiratory position, which leaves the lungspassively during expiration in the course of quiet breathing. As shownin FIGS. 112A-C, animals treated with Budesonide alone showed no changein tidal volumes immediately after the challenge. However, RDC1676-03alone had a significant stimulatory effect on tidal volume even at thisearly time point. And again, RDC1676-03 in combination with Budesonide(both 500 and 750 ug/kg) had an even more pronounced effect on Tidalvolume measurements at this time point. Six hours after the challenge,RDC1676-03 alone was sufficient to cause a significant increase in tidalvolume and addition of Budesonide to the treatment regimen either aloneor in combination had no added effect on tidal volume. Any effectobserved at these earlier time points were, however, lost by the 24hours time point.

Taken together, these data demonstrate that RDC1676-03 alone or incombination with Budesonide provided significant relief to airwayinflammation as evidenced by increase in tidal volume and decrease inPenh values at 6 hours post challenge.

Cytokine Analysis:

To analyze the mechanism of the effects seen on the above discussedphysiological parameters, a number of pro as well as anti-inflammatorycytokines were measured in blood samples collected at 6 and 24 hoursafter the challenge, immediately following the physiologicalmeasurements.

FIGS. 113A and 113B clearly demonstrate that Rev 60 (or RDC1676-03)alone lowered the blood level of eotaxin significantly at both 6 and 24hours post challenge. Budesonide 750 ug/kg also reduced the bloodeotaxin levels at both of these time points, while Budesonide 250 ug/kgonly had a notable effect at the later time point. However, the testsolution Rev 60 alone showed effects that are significantly more potent(in reducing blood eotaxin levels) than both concentrations ofBudesonide, at both time points. Eotaxin is a small C—C chemokine knownto accumulate in and attract eosinophils to asthmatic lungs and othertissues in allergic reactions (e.g., gut in Crohn's disease). Eotaxinbinds to a G protein coupled receptor CCR3. CCR3 is expressed by anumber of cell types such as Th2 lymphocytes, basophils and mast cellsbut expression of this receptor by Th2 lymphocyte is of particularinterest as these cells regulate eosinophil recruitment. Several studieshave demonstrated increased production of eotaxin and CCR3 in asthmaticlung as well as establishing a link between these molecules and airwayhyperresponsiveness (reviewed in Eotaxin and the attraction ofeosinophils to the asthmatic lung, Dolores M Conroy and Timothy JWilliams Respiratory Research 2001, 2:150-156). It is of particularinterest to note that these studies completely agree with the results inFIGS. 109 and 110 on eosinophil counts.

Taken together these results strongly indicate that treatment withRDC1676-03 alone or in combination with Budesonide can significantlyreduce eosinophil total count and % in blood 24 hours after theovalbumin challenge. This correlates with a significant drop in eotaxinlevels in blood observed as early as 6 hours post challenge.

Blood levels of two major key anti-inflammatory cytokines, IL10 andInterferon gamma are also significantly enhanced at 6 hours afterchallenge as a result of treatment with Rev 60 alone or in combinationwith Budesonide. FIGS. 113C and 113D show such effects on Interferongamma and IL10, respectively. It is evident from these figures that Rev60 alone or Rev 60 in combination with Budesonide 250 ug/kgsignificantly increased the blood level of IL10 in the challengedanimals up to 6 hrs post challenge. Similarly, Rev 60 alone or incombination with Budesonide 250 or 750 ug/kg significantly increased theblood level of IFN gamma at 6 hours post challenge. Increase in theseanti-inflammatory cytokines may well explain, at least in part, thebeneficial effects seen on physiological respiratory parameters seen 6hours post challenge. The effect on these cytokines was no longerobserved at 24 hour post challenge (data not shown).

Rantes or CCL5 is a cytokine expressed by circulating T cells and ischemotactic for T cells, eosinophils and basophils and has an activerole in recruiting leukocytes into inflammatory sites. Rantes alsoactivates eosinophils to release, for example, eosinophilic cationicprotein. It changes the density of eosinophils and makes them hypodense,which is thought to represent a state of generalized cell activation. Italso is a potent activator of oxidative metabolism specific foreosinophils.

As shown in FIG. 114, systemic levels of Rantes was reducedsignificantly at 6 hours, but not at 24 hours post challenge in animalstreated with Rev 60 alone or in combination of Budesonide 250 or 750ug/kg. Once again, there is a clear synergistic effect of Budesonide 750ug/kg and Rev 60 that is noted in this set of data. A similar downwardtrend was observed for a number of other pro-inflammatory cytokines,such as KC or IL8, MCP3 μL1b, GCSF, TGFb as well as NGF, observed eitherat 6 or at 24 hours post challenge, in animals treated with Rev60 aloneor in combination with Budesonide.

Example 18 The Inventive Therapeutic Fluids have Substantial Utility forModulating Intercellular Tight Junctions

According to particular aspects, the inventive diffuser processedtherapeutic fluids have substantial utility for modulating intercellulartight junctions, including those relating with pulmonary and systemicdelivery and bioavailability of polypeptides, including the exemplarypolypeptide salmon calcitonin (sCT).

Example Overview. Salmon calcitonin (sCT) is a 32 amino acid peptidewith a molecular weight of 3,432 Daltons. Pulmonary delivery ofcalcitonin has been extensively studied in model systems (e.g., rodentmodel systems, rat model systems, etc) to investigate methods to enhancepulmonary drug delivery (e.g., intratracheal drug delivery). Accordingto particular exemplary aspects, the inventive diffuser processedtherapeutic fluid has substantial utility for modulating (e.g.,enhancing) intercellular tight junctions, for example those associatedwith pulmonary and systemic delivery and bioavailability of sCT in a ratmodel system.

Methods:

Intratracheal drug delivery. According to particular embodiments, sCT isformulated in the inventive therapeutic fluid and administered to ratsusing an intratracheal drug delivery device. In certain aspects, a PennCentury Micro-Sprayer device designed for rodent intratracheal drugdelivery is used, allowing for good lung delivery, but, as appreciatedin the art, with relatively low alveolar deposition resulting in poorsystemic bioavailability of peptides. According to particular aspects,this art-recognized model system was used to confirm that the inventivediffuser processed therapeutic fluid has substantial utility formodulating (e.g., enhancing) intercellular tight junctions, includingthose associated with pulmonary and systemic delivery andbioavailability of polypeptides.

Animal groups and dosing. In certain aspects, rats are assigned to oneof 3 groups (n=6 per group): a) sterile saline; b) base solution withoutO₂ enrichment (‘base solution’); or c) inventive diffuser processedtherapeutic fluid (‘inventive enriched based solution’). The inventiveenriched based solution is formed, for example by infusing oxygen in0.9% saline. Preferably, the base solution comprises about 0.9% salineto minimize the potential for hypo-osmotic disruption of epithelialcells. In certain embodiments, sCT is separately reconstituted in thebase solution and the inventive enriched based solution and therespective solutions are delivered to respective animal groups byintratracheal instillation within 60 minutes (10 μg sCT in 200 μL peranimal).

Assays. In particular aspects, blood samples (e.g., 200 μl) arecollected and placed into EDTA coated tubes prior to dosing and at 5,10, 20, 30, 60, 120 and 240 minutes following dosing. Plasma isharvested and stored at ≦−70° C. until assayed for sCT using an ELISA.

For Agilant gene array data generation, lung tissue was isolated andsubmerged in TRI Reagent (TR118, Molecular Research Center, Inc.).Briefly, approximately 1 mL of TRI Reagent was added to 50-100 mg oftissue in each tube. The samples were homogenized in TRI Reagent, usingglass-Teflon™ or Polytron™ homogenizer. Samples were stored at −80° C.

Results:

Enhancement of tight junctions. FIG. 84 shows that RDC1676-01 (sterilesaline processed through the instant proprietary device with additionaloxygen added; gas-enriched electrokinetically generated fluid (Rev) ofthe instant disclosure) decreased systemic delivery and bioavailabilityof sCT. According to particular aspects, the decreased systemic deliveryresults from decreased adsorption of sCT, most likely resulting fromenhancement of pulmonary tight junctions. RDC1676-00 signifies sterilesaline processed according to the presently disclosed methods, butwithout oxygenation.

Additionally, according to particular aspects, tight junction relatedproteins were upregulated in lung tissue. FIGS. 85-89 show upregulationof the junction adhesion molecules JAM 2 and 3, GJA1, 3, 4 and 5(junctional adherins), OCLN (occludin), claudins (e.g., CLDN 3, 5, 7, 8,9, 10), TJP1 (tight junction protein 1), respectively.

Example 19 The Inventive Therapeutic Fluids have Substantial Utility forModulating Nitric Oxide Levels

According to particular aspects, the inventive diffuser processedtherapeutic fluids have substantial utility for modulating nitric oxidelevels, and/or related enzymes. FIGS. 90-94 show data obtained fromhuman foreskin keratinocytes exposed to RDC1676-01 (sterile salineprocessed through the instant proprietary device with additional oxygenadded; gas-enriched electrokinetically generated fluid (Rev) of theinstant disclosure) showing up-regulation of NOS1 and 3, and Nostrin,NOS3. By contrast, data obtained from rat lung tissue (tissue of aboveExample entitled “Cytokine Expression”) shows down regulation of NOS2and 3, Nostrin and NOS1AP with Rev (FIGS. 93, 94).

Example 20 Localized Electrokinetic Effects (Voltage/Current) wereDemonstrated Using a Specially Designed Mixing Device ComprisingInsulated Rotor and Stator Features

In this Example, feature-localized electrokinetic effects(voltage/current) were demonstrated using a specially designed mixingdevice comprising insulated rotor and stator features.

Overview. As discussed in detail herein above under “Double LayerEffect” (see also FIGS. 26 and 28) The mixing device 100 may beconfigured to create the output material 102 by complex and non-linearfluid dynamic interaction of the first material 110 and the secondmaterial 120 with complex, dynamic turbulence providing complex mixingthat further favors electrokinetic effects. According to particularaspects, the result of these electrokinetic effects may be presentwithin the output material 102 as charge redistributions and redoxreactions, including in the form of solublized electrons that arestabilized within the output material.

In addition to general surface-related double layer effects in themixing chamber, Applicants additionally reasoned that localizedelectrokinetic effects may be imparted by virtue of the feature-inducedmicrocavitation and fluid acceleration and deceleration in the vicinityof the features. The studies of this Example were thus performed tofurther investigate and confirm said additional electrokinetic aspects.

Materials:

A test device similar to the inventive mixing devices described hereinwas constructed, comprising a stainless steel rotor 12 having twofeatures 18 (disposed at 180 degrees), and a stator 14 with a singlefeature 16 positioned to be rotationally opposable to the rotor features18 and stator features 16. Significantly, the rotor and stator features,in each case, are insulated from the respective rotor and stator bodies(FIG. 95). The device was machined to provide for a consistentrotor:stator gap 20 of 0.020 inches to conform with the devicesdisclosed elsewhere herein. There is a rotating contact (not shown) atthe end of the rotor shaft (not shown) that provides an electrical pathfor the rotor surface and for the insulated rotor features. Likewise thestator has a similar insulated feature 16 (FIG. 95), wherein the statorinner surface and the insulated stainless steel feature are connected torespective contacts on the stator exterior.

A operational amplifier (OpAmp) circuit (M) 22 is connected between thecontacts. The operational amplifier (OpAmp) circuit was constructed toprovide for collection of very low voltage measurements by takingadvantage of the high input impedance of such amplifiers. The outputs ofthe OpAmp are fed to the inputs of an oscilloscope (e.g., a batterypowered laptop running an oscilloscope application with a Pico Scope3000™).

To eliminate the introduction of any ambient noise (e.g., RF radiationfrom wireless network signals and from the 60 Hz power line) duringtesting of the device, a fine copper mesh, RF-shielded compartment(approx. three by four by four feet) was constructed to provide aFaraday cage. This configuration provided for excellent signal to noiseratios during experimental testing, as interfering signals from 60 Hz ACnoise (e.g., of approximately two volts) and high frequency RF wasreduced well below the signals of interest. Using a battery poweredlaptop running an oscilloscope application with a Pico Scope 3000enabled detection of the 30 mV signals (as in FIG. 96) created by thefeatures of the test device. In addition, a variable speed DC motor waspositioned outside the Faraday cage and coupled to the rotatable testdevice via a non-metallic shaft to effectively isolate the motor noiseaway from the test device.

Methods:

The OpAmp circuit was used to measure voltage potential between thecontacts connecting the stator inner surface 12 and the insulated statorfeature 16. With the particular circuit arrangement, only a potentialwas measured. The rotational speed of the device could be varied betweenabout 700 to about 2800 rpm (with the data of FIG. 96 being measuredwith the device running at about 1800 rpm).

To avoid any extraneous voltage generation due to a pump or peristalticpump, fluid flow through the device was accomplished using inertnitrogen or air or argon acting on fluid in tanks connected to thedevice. There was no perceptible voltage contribution from the flowmechanism, and typically air was used as the pumping force to providefor fluid flow through the device.

Fluid flow rate through the device was about 1 L/min.

An initial set of non-rotational experiments was conducted by directingfluid flow through the device chamber but without rotation of the rotorin order to assess the presence of any voltage between the stator body12 and the isolated feature 16. Separate experiments were conducted forboth flow directions.

An additional set of rotational experiments was then conducted with thesame fluid flow rate, and with the device rotor rotating at variousspeeds from about 300 to about 1800 rpm. For any given experiment, theflow rate and rotational speed were held constant.

Results:

With respect to the non-rotational experiments, with fluid flowingthrough the device in either direction without any rotor rotation therewas only a barely perceptible voltage (e.g., 1 to 2 mV)) between thebody of the stator and the insulated feature.

With respect to the rotational experiments, and with reference to FIG.96, it can be seen that voltage pulses (potential pulses), temporallycorrelating (in this case at about 1800 rpm) with rotational alignmentof opposing rotor stator features, were measurable with the OpAmp in theoperating test device. Moreover, such periodic voltage pulses,correlating with feature alignments, could be observed over a range fromabout 250 or 300 rpm to about 1800. Additionally, with or without fluidflow, such voltage pulses were observed in the rotational experiments aslong as the cavity/fluid chamber of the device was filled with fluid.According to particular aspects, and without being bound by mechanism,rapid, violent compression (e.g., cavitation), acceleration anddeceleration of fluid flow in the vicinity of the repetitiverotationally aligned features created the respective local voltagepulses that correlate exactly with the rotational period, providing, atleast in part, for electrokinetically generated fluid according to thepresent invention. Additional experiments revealed that the amplitude(peak shape and height) of the voltage pulses increased with increasingrotational velocity, being initially observable at about 250 to 300 rpmin this particular test device, and increasing up to at least about 2800rpm. The magnitude of the violent acceleration and deceleration, etc.,of fluid flow in the vicinity of the rotationally aligned features wouldbe expected to generally increase with increasing rotational velocity;at least until a maximum was reached reflecting physical limits imposedby the geometry, configuration and/or flow rate of the device. Accordingto additional aspects, because localized voltage spikes are present,localized current flow (e.g., current pulses) is generated in thevicinity of the features, providing, at least in part, forelectrokinetically generated fluid according to the present invention(e.g., without being bound by mechanism, providing for electrochemicalreactions as discussed elsewhere herein).

According to additional aspects, and without being bound by mechanism,such feature-localized effects (e.g., voltage pulses and current and/orcurrents pulses) contribute to generation of the electrokineticallygenerated fluids in combination with more general surface-related doublelayer and streaming current effects discussed elsewhere herein aboveunder “Double Layer Effect” (see also FIGS. 26 and 28).

Example 21 Relative to Non-Electrokinetically Generated Control Fluids,the Inventive Electrokinetically Generated Fluids were Shown toDifferentially Affect Line Widths in ¹³C NMR Analysis of the DissolvedSolute α,α-Trehalose

Overview. Applicants data disclosed elsewhere herein support utility andmechanism wherein the inventive electrokinetically generated fluidsmediate regulation or modulation of intracellular signal transduction bymodulation of at least one of cellular membranes, membranepotential/conductance, membrane proteins (e.g., membrane receptors suchas G protein coupled receptors), calcium dependant cellular signalingsystems, and intercellular junctions (e.g., tight junctions, gapjunctions, zona adherins and desmasomes). Specifically, using a varietyof art-recognized biological test systems and assays, Applicants datashows, relative to control fluids, differential effects of the inventivefluid on, for example: regulatory T cell proliferation; cytokine andprotein levels (e.g., IL-10, GITR, Granzyme A, XCL1, pStat5, and Foxp3,tyrptase, tight junction related proteins, TSLP receptor, MMP9, etc.);binding of Bradykinin ligand with the Bradykinin B2 receptor; expressionof TSLP receptor, whole cell conductance; etc. Moreover, the Diphtheriatoxin (DT390) effects shown herein indicate that beta blockade (beta 2adrenergic receptor), and/or GPCR blockade and/or Ca channel blockadeaffects the activity of the electrokinetically generated fluids on, forexample, Treg and PBMC function.

Taken together these effects indicate that the inventiveelectrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids, but also that they provide fornovel compositions and substantial utilities such as those presentlydisclosed and claimed herein.

In this Example. Applicants have in this Example performed nuclearmagnetic resonance (NMR) studies to further characterize the fundamentalnature of the inventive electrokinetically generated fluids.Specifically, Applicants have analyzed the ¹³C NMR spectra ofα,α-Trehalose dissolved in the electrokinetically generated fluid,compared to dissolution in non-electrokinetically generated fluid.Trehalose (shown below with carbons numbered for reference) is acosmotrophic solute and is known, for example to protect against proteindenaturation, membrane desiccation, organism viability upon freezing,etc. Applicants, given the data summarized above, reasoned thatα,α-Trehalose might provide an effective tool to further probe theproperties/structure of the inventive electrokinetically generatedfluids. Applicants reasoned that NMR-related ‘chemical shifts’ andeffects on ‘line widths’ could be used to assess properties of theinventive fluids. For these studies, a non-superoxygenated inventiveelectrokinetically generated fluid (referred to herein as “Solas”) wasemployed to minimize the possibility that paramagnetic impurities, suchas dissolved oxygen, might act to counter or otherwise mask the effectsbeing analyzed.

Materials and Methods:

Solution Preparation. The Phosphate (sodium salt) and D-(+)-Trehalosedihydrate (T9531-10G, reduced metal content) and 99.9% D2O containing 1%DSS were purchased from Sigma. The “Normal Saline” is 0.9% SodiumChloride, pH 5.6 (4.5-7.0), from Hospira. The 0.25 M α,α-Trehalosesolutions were prepared by dissolving 0.949 g trehalose into 965 μLNormal Saline and 35 mL Phoshate Buffered Saline (100 mM PhosphateBuffer in 0.9% NaCl preparted in such a way that when 35 μL of thisbuffer are added to 1.0 mL trehalose solution the pH becomes 6.93).

Nuclear Magnetic Resonance Spectra Collection. Spectra were collected atthe University of Washington NMR facility using either an 500 MHz or 300MHz Bruker Avance series instrument fitted with a Bruker BBO: X {1H}probe and running XWINNMR 3.5. ¹³C NMR spectra were collected at 125.7MHz or 75.46 MHz using a 14000 Hz or 7900 Hz sweep width using 64K or128K data points and 128 or 256 scans. The resulting FIDs werezero-filled twice and processed with a 1.0 Hz line broadening factor.Temperature was controlled using the Bruker Biospin Variable Temperatureunit. External deuterium locking was employed by placing 99.9% D2O+1%DSS+a trace of acetone in a coaxial NMR insert tube, purchased fromWilmad. The NMR data was processed using the iNMR software v. 2.6.4 fromMestrelab Research.

Results:

Sample Spectra. FIG. 97A-C shows expansions of six ¹³C-NMR spectraoverlaid on top of each other such that the DSS signals line up at −2.04ppm. The DSS signals are shown at the far right of the figure, and theacetone methyl signal is shown near 30.9 ppm. The remaining signalscorrespond to the 6 carbons of trehalose as shown in the α,α-Trehalosestructure above. As can be seen, the carbon signals in the Solassolutions show small chemical shifts (generally upfield) compared to thecontrol solutions.

Line Width Measurements. TABLE 9 below shows the measured ¹³C NMR linewidths for the six carbons of trehalose and the methyl carbon of acetoneat 3 different temperatures for Solas Saline (an inventiveelectrokinetically generated fluid). The corresponding Normal Salinesamples represent non-electrokinetic control solutions at eachtemperature. In the Solas solutions, the line widths are significantlydifferent from the line widths in the control solution for each carbonatom. The smaller linewidths in the Solas solutions at lowertemperatures likely result from a faster tumbling rate of the trehalosemolecule as a whole (including any solvated water molecules) compared tothe control solutions.

TABLE 9 ¹³C NMR Line Widths for α,α-Trehalose in Solas & NormalSaline^(a,b) Test Fluid (Temp. degrees K) C-1 C-2 C-3 C-4 C-5 C-6Acetone Solas (277) 8.4 8.22 8.3 8.15 8.3 11.1 5.1 Normal (269.9) 15.416.1 15.8 14.9 15.4 21.7 5.1 Solas (293) 9.52 8.7 9.28 9 8.9 11.25 5.63Normal (292.9) 10.33 10.23 10.23 9.93 10.23 13.13 5.63 Solas (310) 2.282.03 2.18 2.19 2 2.55 0.67 Normal (309.9) 1.17 0.99 1.1 1.02 0.97 1.420.67 ^(a)1.0 Hz was subtracted from all line width values due to the 1.0Hz line broadening used during processing. In addition, line widthvalues were normalized relative to the acetone signal in the externalreference tube in order to compensate for magnetic fieldinhomogeneities. This was done by subtracting from the Normal Salineline widths the amount by which the acetone peak was broadened in thecorresponding Solas Saline spectra. ^(b)Error in line width measurementsestimated to be within +/−0.30 Hz

The ¹³C NMR line widths for α,α-Trehalose in Solas and normal saline, ineach case normalized with respect to the Acetone line, are showngraphically in FIG. 97A. In conclusion, the NMR data for ¹³C NMR linewidths for α,α-Trehalose in Solas and normal saline indicate that thereis a property of the inventive solution which alters solute tumbling.

Taken together with the biological activities summarize above andelsewhere herein, these ¹³C NMR line width effects indicate that theinventive electrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids in terms of solute interactions, butalso that they provide for novel compositions and substantial utilitiessuch as those presently disclosed and claimed herein.

Example 22 Relative to Non-Electrokinetically Generated Control Fluids,the Inventive Electrokinetically Generated Fluids Produced DifferentialSquare Wave Voltametry Profiles And Displayed Unique ElectrochemicalProperties Under Stripping Polarography

Overview. Applicants' data disclosed elsewhere herein support utilityand mechanism wherein the inventive electrokinetically generated fluidsmediate regulation or modulation of intracellular signal transduction bymodulation of at least one of cellular membranes, membranepotential/conductance, membrane proteins (e.g., membrane receptors suchas G protein coupled receptors), calcium dependant cellular signalingsystems, and intercellular junctions (e.g., tight junctions, gapjunctions, zona adherins and desmasomes). Specifically, using a varietyof art-recognized biological test systems and assays. Applicants datashows, relative to control fluids, differential effects of the inventivefluid on, for example: regulatory T cell proliferation; cytokine andprotein levels (e.g., IL-10, GITR, Granzyme A, XCL1, pStat5, and Foxp3,tyrptase, tight junction related proteins, TSLP receptor, MMP9, etc.);binding of Bradykinin ligand with the Bradykinin B2 receptor; expressionof TSLP receptor, whole cell conductance; etc. Moreover, the Diphtheriatoxin (DT390) effects shown herein indicate that beta blockade (beta 2adrenergic receptor), and/or GPCR blockade and/or Ca channel blockadeaffects the activity of the electrokinetically generated fluids on, forexample, Treg and PBMC function.

Taken together these effects indicate that the inventiveelectrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids, but also that they provide fornovel compositions and substantial utilities such as those presentlydisclosed and claimed herein.

In this Example. Applicants have, in this Example, performed voltametrystudies to further characterize the fundamental nature of the inventiveelectrokinetically generated fluids. Voltametry is frequently used todetermine the redox potential or measure kinetic rates and constants offluids. The common characteristic of all voltametric methods is thatthey involve the application of a potential to an electrode and theresultant current flowing is monitored through an electrochemical cell.The applied potential produces a change in the concentration of anelectroactive species at the electrode surface by electrochemicallyreducing or oxidizing the species.

Specifically, Applicants have utilized voltametric methods (i.e., squarewave voltametry and stripping polarography) to further characterizefundamental differences between control saline fluid and the inventiveelectrokinetically generated test fluids (e.g., Solas and Revera).Applicants, given the biological and membrane effects data summarizedabove, reasoned that square wave voltametry and stripping polarographywould provide an effective means to further characterize the uniqueproperties of the inventive electrokinetically generated fluids.

Applicants further reasoned that differences in current at specificvoltages, production of different concentrations of an electroactiveredox compound, creation of new redox compounds, and possession ofunique electrochemical properties could be used to assess andcharacterize properties of the inventive fluids. For these studies, botha superoxygenated electrokinetically generated fluid (Revera), and anon-superoxygenated inventive electrokinetically generated fluid (Solas)were used.

Materials and Methods:

Materials and Solution Preparation. The experiments were conducted on anEG & G SMDE 303A polarographer (Princeton Applied Research). Theelectrolyte, NaOH, used in the square wave voltametry experiment, waspurchased from Sigma. A 10 mL sample of the inventive fluid solution wasprepared by adding 100 μL of NaOH to 9.9 mL of Revera Saline to make a0.18 molar solution. With regards to the stripping polarographyexperiment, no extra electrolyte was utilized.

Square Wave Voltametry. As stated above, voltametry is used to determinethe redox potential or measure kinetic rates and constants in fluids. Inthe square wave voltametry experiment, a potential of 0.0 toapproximately −1.75 V was applied to an electrode and the resultantcurrent flowing through the electrochemical cell was monitored.

Stripping Polarography. The stripping polarography method is similar tothe square wave voltametry method. However, no electrolyte was utilizedas stated above and also involved a pre-step. In the pre-step, thestatic mercury drop electrode was held for 30 seconds at −1.1 V toamalgamate any compounds whose reduced form was soluble in mercury.Then, the potentials between −1.1 V and 0.0 V were scanned and theresultant current flowing through the electrochemical cell wasmonitored. A linear scan into the negative potentials on this amalgamprovided a sensitive measurement of these compounds.

Results:

Square Wave Voltametry. As evident from FIG. 98, the current profiles at−0.14V, −0.47V, −1.02V and −1.36V differ between the various testedagents. According to particular aspects, the differences in currentgenerated at the various specific voltages indicate at least one of adifferent concentration of an electroactive redox compound and/or a newor unique electroactive redox compound, and/or a change in thediffusion-limiting electrical double layer surrounding the mercury drop.

Stripping Polarography. FIG. 99 shows that the inventiveelectrokinetically generated fluids, Revera and Solas, show uniquespectra with pronounced peaks at −0.9 volts that are not present in thenon-electrokinetically generated blank and saline control fluids.Additionally, the spectra of the non-electrokinetically generated blankand saline control fluids show characteristic peaks at −0.19 and −0.3volts that are absent in the spectra for the electrokineticallygenerated Solas and Revera fluids.

According to particular aspects, therefore, these results show uniqueelectrochemical properties of the inventive electrokinetically generatedSolas and Revera fluids compared to non-electrokinetically generatedSaline control fluid. According to additional aspects, the resultsindicate the presence or generation of at least one of a differentconcentration of an electroactive redox compound and a new and/or uniqueelectroactive redox compound in electrokinetically generated versusnon-electrokinetically generated fluids.

On top of the various biological data presented elsewhere herein, thisdifferential voltametry data, particularly when considered along withthe differential effects on whole cell conductance, ¹³C NMR line-widthanalysis, and the mixing device feature-localized effects (e.g., voltagepulses and current and/or currents pulses) indicate that the inventiveelectrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids, but also provide for novelcompositions and substantial utilities such as those presently disclosedand claimed herein.

Example 23 Patch Clamp Analysis Conducted on Bronchial Epithilial Cells(BEC) Perfused with Inventive Electrokinetically Generated Fluid(RNS-60) Revealed that Exposure to RNS-60 Resulted in a Decrease inWhole Cell Conductance, and Stimulation with a cAMP Stimulating“Cocktail”, which Dramatically Increased the Whole-Cell Conductance, andAlso Increased the Drug-Sensitive Portion of the Whole-Cell Conductance,which was Ten-Times Higher than that Observed Under Basal Conditions

In this Example, patch clamp studies were performed to further confirmthe utility of the inventive electrokinetically generated fluids tomodulate intracellular signal transduction by modulation of at least oneof membrane structure, membrane potential or membrane conductivity,membrane proteins or receptors, ion channels, and calcium dependantcellular messaging systems.

Overview. As shown in Example 14 above (e.g., FIG. 75, showingStabilization of Bradykinin binding to the B2 receptor using Bio-LayerInterferometry biosensor, Octet Rapid Extended Detection (RED)(forteBio™)), Bradykinin binding to the B2 receptor was concentrationdependent, and binding affinity was increased in the electrokineticallygenerated fluid (e.g., Rev; gas-enriched electrokinetically generatedfluid) of the instant disclosure compared to normal saline.Additionally, as shown in Example 15 in the context of T-regulatorycells stimulated with particulate matter (PM), the data showed adecreased proliferation of T-regulatory cells in the presence of PM andRev relative to PM in control fluid (no Rev, no Solas) (FIG. 76),indicating that the inventive electrokinetically generated fluid Revimproved regulatory T-cell function; e.g., as shown by relativelydecreased proliferation in the assay. Moreover, exposure to theinventive fluids resulted in a maintained or only slightly decreasedproduction of IL-10 relative to the Saline and Media controls (no PM).Likewise, in the context of the allergic asthma (AA) profiles ofperipheral blood mononuclear cells (PBMC) stimulated with particulatematter (PM), the data showed that exposure to the fluids of the instantdisclosure (“PM+Rev”) resulted in significantly lower tryptase levelssimilar to those of the Saline and Media controls. Additionally, theDiphtheria toxin (DT390) effects shown in Example 15 and FIGS. 76-83,indicate that beta blockade, GPCR blockade and Ca channel blockadeaffects the activity of the electrokinetically generated fluids on Tregand PBMC function. Furthermore, the data of Example 18 shows that,according to additional aspects, upon expose to the inventive fluids,tight junction related proteins were upregulated in lung tissue. FIGS.85-89 show upregulation of the junction adhesion molecules JAM 2 and 3,GJA1, 3, 4 and 5 (junctional adherins), OCLN (occludin), claudins (e.g.,CLDN 3, 5, 7, 8, 9, 10), TJP1 (tight junction protein 1), respectively.

Patch clamp studies were performed to further investigate and confirmsaid utilities.

Materials and Methods:

The Bronchial Epithelial line Calu-3 was used in Patch clamp studies.Calu-3 Bronchial Epithelial cells (ATCC #HTB-55) were grown in a 1:1mixture of Ham's F12 and DMEM medium that was supplemented with 10% FBSonto glass coverslips until the time of the experiments. In brief, awhole cell voltage clamp device was used to measure effects on Calu-3cells exposed to the inventive electrokinetically generated fluids(e.g., RNS-60; electrokinetically treated normal saline comprising 60ppm dissolved oxygen; sometimes referred to as “drug” in this Example).

Patch clamping techniques were utilized to assess the effects of thetest material (RNS-60) on epithelial cell membrane polarity and ionchannel activity. Specifically, whole cell voltage clamp was performedupon the Bronchial Epithelial line Calu-3 in a bathing solutionconsisting of: 135 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, and 10mM HEPES (pH adjusted to 7.4 with N-methyl D-Glucamine). Basal currentswere measured after which RNS-60 was perfused onto the cells.

More specifically, patch pipettes were pulled from borosilicate glass(Garner Glass Co, Claremont, Calif.) with a two-stage Narishige PB-7vertical puller and then fire-polished to a resistance between 6-12Mohms with a Narishige MF-9 microforge (Narishige International USA,East Meadow, N.Y.). The pipettes were filled with an intracellularsolution containing (in mM): 135 KCl, 10 NaCl, 5 EGTA, 10 Hepes, pH wasadjusted to 7.4 with NMDG (N-Methyl-D-Glucamine).

The cultured Calu-3 cells were placed in a chamber containing thefollowing extracellular solution (in mM): 135 NaCl, 5 KCl, 1.2 CaCl2,0.5 MgCl2 and 10 Hepes (free acid), pH was adjusted to 7.4 with NMDG.

Cells were viewed using the 40×DIC objective of an Olympus IX71microscope (Olympus Inc., Tokyo, Japan). After a cell-attached gigasealwas established, a gentle suction was applied to break in, and to attainthe whole-cell configuration. Immediately upon breaking in, the cell wasvoltage clamped at −120, −60, −40 and 0 mV, and was stimulated withvoltage steps between +100 mV (500 ms/step). After collecting thewhole-cell currents at the control condition, the same cell was perfusedthrough bath with the test fluid comprising same exatracelluar solutesand pH as for the above control fluid, and whole-cell currents atdifferent holding potentials were recorded with the same protocols.

Electrophysiological data were acquired with an Axon Patch 200Bamplifier, low-pass filtered at 10 kHz, and digitized with 1400ADigidata (Axon Instruments, Union City, Calif.). The pCLAMP 10.0software (Axon Instruments) was used to acquire and to analyze the data.Current (I)-to-voltage (V) relationships (whole cell conductance) wereobtained by plotting the actual current value at approximately 400 msecinto the step, versus the holding potential (V). The slope of the I/Vrelationship is the whole cell conductance.

Drugs and Chemicals. Whenever indicated, cells were stimulated with acAMP stimulatory cocktail containing 8-Br-cAMP (500 mM), IBMX(isobutyl-1-methylxanthie, 200 mM) and forskolin (10 mM). The cAMPanalog 8-Br-cAMP (Sigma Chem. Co.) was used from a 25 mM stock in H2Osolution. Forskolin (Sigma) and IBMX (Sigma) were used from a DMSOsolution containing both 10 mM Forskolin and 200 mM IBMX stock solution.

Patch Clamp Results:

FIG. 100 shows whole-cell currents under basal (no cAMP) conditions,with a protocol stepping from zero mV holding potential to +/−100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance, obtainedfrom the current-to-voltage relationships is highly linear under bothconditions, and reflects a modest, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug(inventive electrokinetically generated fluid) is also linear, and thereversal potential is near zero mV. There is a decrease in the wholecell conductance under hyperpolarizing conditions.

FIG. 101 shows whole-cell currents under basal conditions, with aprotocol stepping from −40 mV holding potential to ±100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance obtained fromthe current-to-voltage relationships is highly linear under bothconditions, and reflects a modest, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug(inventive electrokinetically generated fluid) is also linear, and thereversal potential is near zero mV. Values are comparatively similar tothose obtained with the zero mV protocol.

FIG. 102 shows whole-cell currents under basal conditions, with aprotocol stepping from −60 mV holding potential to ±100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance obtained fromthe current-to-voltage relationships is highly linear under bothconditions, and reflects a minor, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug isalso linear, and the reversal potential is near zero mV. Values arecomparatively similar to those obtained with the zero mV protocol.

FIG. 103 shows whole-cell currents under basal conditions, with aprotocol stepping from −120 mV holding potential to ±100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance obtained fromthe current-to-voltage relationships is highly linear under bothconditions, and reflects a minor, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug isalso linear, and the reversal potential is near zero mV. Values arecomparatively similar to those obtained with the zero mV protocol.

FIG. 104 shows whole-cell currents under cAMP-stimulated conditions,obtained with protocols stepping from various holding potentials to ±100mV. Representative tracings are the average of n=5 cells. The tracingson the left are the control, followed by the whole-cell tracings aftercAMP stimulation, followed by perfusion with the drug-containingsolution. The tracings on the right are the composite delta obtained bysubtraction of the test average values in drug +cAMP, from those undercontrol conditions (cAMP alone). The tracings on the Top are thoseobtained from voltage protocol at zero mV, and the ones below, at −40mV. The whole-cell conductance obtained from the current-to-voltagerelationships is highly linear under all conditions, and reflects achange in conductance due to the test conditions.

FIG. 105 shows whole-cell currents under cAMP-stimulated conditions,obtained with protocols stepping from various holding potentials to ±100mV. Representative tracings are the average of n=5 cells. The tracingson the left are the control, followed by the whole-cell tracings aftercAMP stimulation, followed by perfusion with the drug-containingsolution. The tracings on the right are the composite delta obtained bysubtraction of the test average values in drug +cAMP, from those undercontrol conditions (cAMP alone). The tracings on the Top are thoseobtained from voltage protocol at −60 mV, and the ones below, at −120mV. The whole-cell conductance, obtained from the current-to-voltagerelationships, is highly linear under all conditions, and reflects achange in conductance due to the test conditions.

FIG. 106 shows the effect of holding potential on cAMP-activatedcurrents. The effect of the drug (the inventive electrokineticallygenerated fluids; RNS-60; electrokinetically treated normal salinecomprising 60 ppm dissolved oxygen) on the whole-cell conductance wasobserved under different voltage protocols (0, −40, −60, −120 mV holdingpotentials). Under basal conditions, the drug-sensitive whole-cellcurrent was identical at all holding potentials (voltage-insensitivecontribution, Top Left panel). In the cAMP-activated conditions,however, the drug-sensitive currents were much higher, and sensitive tothe applied voltage protocol. The current-to-voltage relationships arehighly nonlinear. This is further observed in the subtracted currents(Bottom panel), where the contribution of the whole cell conductance atzero mV was further subtracted for each protocol (n=5).

Summary of Example. According to particular aspects, therefore, the dataindicate that there is a modest but consistent effect of the drug (theinventive electrokinetically generated fluids; RNS-60;electrokinetically treated normal saline comprising 60 ppm dissolvedoxygen) under basal conditions. To enhance the effect of the drug on thewhole-cell conductance, experiments were also conducted by perfusing thedrug after stimulation with a cAMP stimulating “cocktail”, whichdramatically increased the whole-cell conductance. Interestingly, thisprotocol also increased the drug-sensitive portion of the whole-cellconductance, which was ten-times higher than that observed under basalconditions. Additionally, in the presence of cAMP stimulation, the drugshowed different effects with respect to the various voltage protocols,indicating that the electrokinetically generated fluids affect avoltage-dependent contribution of the whole-cell conductance. There wasalso a decrease in a linear component of the conductance, furthersuggesting at least a contribution of the drug to the inhibition ofanother pathway (e.g., ion channel, voltage gated cation channels,etc.).

In particular aspects, and without being bound by mechanism, Applicants'data are consistent with the inventive electrokinetically generatedfluids (e.g., RNS-60; electrokinetically treated normal salinecomprising 60 ppm dissolved oxygen) producing a change either on achannel(s), being blocked or retrieved from the plasma membrane.

Taken together with Applicants' other data (e.g., the data of workingExamples) particular aspects of the present invention providecompositions and methods for modulating intracellular signaltransduction, including modulation of at least one of membranestructure, membrane potential or membrane conductivity, membraneproteins or receptors, ion channels, and calcium dependant cellularsignalling systems, comprising use of the inventive electrokineticallygenerated solutions to impart electrochemical and/or conformationalchanges in membranous structures (e.g., membrane and/or membraneproteins, receptors or other components) including but not limited toGPCRs and/or g-proteins. According to additional aspects, these effectsmodulate gene expression, and may persist, dependant, for example, onthe half lives of the individual messaging components, etc.

Example 24 Patch Clamp Analysis Conducted on Calu-3 Cells Perfused withInventive Electrokinetically Generated Fluids (RNS-60 and Solas)Revealed that (i) Exposure to RNS-60 and Solas Resulted in Increases inWhole Cell Conductance, (ii) that Exposure of Cells to the RNS-60Produced an Increase in a Non-Linear Conductance, Evident at 15 MinIncubation Times, and (iii) that Exposure of Cells to the RNS-60Produced an Effect of RNS-60 Saline on Calcium Permeable Channels

Overview. In this Example, patch clamp studies were performed to furtherconfirm the utilities, as described herein, of the inventiveelectrokinetically generated saline fluids (RNS-60 and Solas), includingthe utility to modulate whole-cell currents. Two sets of experimentswere conducted.

The summary of the data of the first set of experiments indicates thatthe whole cell conductance (current-to-voltage relationship) obtainedwith Solas saline is highly linear for both incubation times (15 min, 2hours), and for all voltage protocols. It is however evident, thatlonger incubation (2 hours) with Solas increased the whole cellconductance. Exposure of cells to the RNS-60 produced an increase in anon-linear conductance, as shown in the delta currents (Rev-Solsubtraction), which is only evident at 15 min incubation time. Theeffect of the RNS-60 on this non-linear current disappears, and isinstead highly linear at the two-hour incubation time. The contributionof the non-linear whole cell conductance, as previously observed, wasvoltage sensitive, although present at all voltage protocols.

The summary of data of the second set of experiments indicates thatthere is an effect of the RNS-60 saline on a non-linear current, whichwas made evident in high calcium in the external solution. Thecontribution of the non-linear whole cell conductance, although voltagesensitive, was present in both voltage protocols, and indicates aneffect of RNS-60 saline on calcium permeable channels.

First Set of Experiments (Increase of Conductance; and Activation of aNon-Linear Voltage Regulated Conductance) Methods for First Set ofExperiments:

See EXAMPLE 23 for general patch clamp methods. In the following firstset of experiments, patch clamp studies were performed to furtherconfirm the utility of the inventive electrokinetically generated salinefluids (RNS-60 and Solas) to modulate whole-cell currents, using Calu-3cells under basal conditions, with protocols stepping from either zeromV holding potential, −120 mV, or −60 mV.

The whole-cell conductance in each case was obtained from thecurrent-to-voltage relationships obtained from cells incubated foreither 15 min or two hours, to further confirm the results of EXAMPLE23. In this study, groups were obtained at a given time, for eitherSolas or RNS-60 saline solutions. The data obtained are expressed as themean±SEM whole cell current for 5-9 cells.

Results:

FIGS. 117 A-C show the results of a series of patch clamping experimentsthat assessed the effects of the electrokinetically generated fluid(e.g., RNS-60 and Solas) on epithelial cell membrane polarity and ionchannel activity at two time-points (15 min (left panels) and 2 hours(right panels)) and at different voltage protocols (A, stepping fromzero mV; B, stepping from −60 mV; and C, stepping from −120 mV). Theresults indicate that the RNS-60 (filled circles) has a larger effect onwhole-cell conductance than Solas (open circles). In the experimentsimilar results were seen in the three voltage protocols and at both the15 minute and two-hour incubation time points.

FIGS. 118 A-C show graphs resulting from the subtraction of the Solascurrent data from the RNS-60 current data at three voltage protocols(“Delta currents”) (A, stepping from zero mV; B, stepping from −60 mV;and C, stepping from −120 mV) and the two time-points (15 mins (opencircles) and 2 hours (filled circles)). These data indicated that at the15 minute time-point with RNS-60, there is a non-linearvoltage-dependent component that is absent at the 2 hour time point.

As in previous experiments, data with “Normal” saline gave a veryconsistent and time-independent conductance used as a reference. Thepresent results were obtained by matching groups with either Solas orRNS-60 saline, and indicate that exposure of Calu-3 cells to the RNS-60saline under basal conditions (without cAMP, or any other stimulation),produces time-dependent effect(s), consistent with the activation of avoltage-regulated conductance at shorter incubation times (15 min). Thisphenomenon was not as apparent at the two-hour incubation point. Asdescribed elsewhere herein, the linear component is more evident whenthe conductance is increased by stimulation with the cAMP “cocktail”.Nonetheless, the two-hour incubation time showed higher linearconductance for both the RNS-60 and the Solas saline, and in this case,the RNS-60 saline doubled the whole cell conductance as compared toSolas alone. This evidence indicates that at least two contributions tothe whole cell conductance are affected by the RNS-60 saline, namely theactivation of a non-linear voltage regulated conductance, and a linearconductance, which is more evident at longer incubation times.

Second Set of Experiments (Effect on Calcium Permeable Channels) Methodsfor Second Set of Experiments:

See EXAMPLE 23 for general patch clamp methods. In the following secondset of experiments, yet additional patch clamp studies were performed tofurther confirm the utility of the inventive electrokineticallygenerated saline fluids (RNS-60 and Solas) to modulate whole-cellcurrents, using Calu-3 cells under basal conditions, with protocolsstepping from either zero mV or −120 mV holding potentials.

The whole-cell conductance in each case was obtained from thecurrent-to-voltage relationships obtained from cells incubated for 15min with either saline. To determine whether there is a contribution ofcalcium permeable channels to the whole cell conductance, and whetherthis part of the whole cell conductance is affected by incubation withRNS-60 saline, cells were patched in normal saline after the incubationperiod (entails a high NaCl external solution, while the internalsolution contains high KCl). The external saline was then replaced witha solution where NaCl was replaced by CsCl to determine whether there isa change in conductance by replacing the main external cation. Underthese conditions, the same cell was then exposed to increasingconcentrations of calcium, such that a calcium entry step is made moreevident.

Results:

FIGS. 119 A-D show the results of a series of patch clamping experimentsthat assessed the effects of the electrokinetically generated fluid(e.g., Solas (panels A and B) and RNS-60 (panels C and D)) on epithelialcell membrane polarity and ion channel activity using different externalsalt solutions and at different voltage protocols (panels A and C showstepping from zero mV, whereas panels B and D show stepping from −120mV). In these experiments one time-point of 15 minutes was used. ForSolas (panels A and B) the results indicate that: 1) using CsCl (squaresymbols) instead of NaCl as the external solution, increased whole cellconductance with a linear behavior when compared to the control (diamondsymbols); and 2) CaCl₂ at both 20 mM CaCl₂ (circle symbols) and 40 mMCaCl₂ (triangle symbols) increased whole cell conductance in anon-linear manner. For RNS-60 (panels C and D), the results indicatethat: 1) using CsCl (square symbols) instead of NaCl as the externalsolution had little effect on whole cell conductance when compared tothe control (diamond symbols); and 2) CaCl₂ at 40 mM (triangle symbols)increased whole cell conductance in a non-linear manner.

FIGS. 120 A-D show the graphs resulting from the subtraction of the CsClcurrent data (shown in FIG. 119) from the 20 mM CaCl₂ (diamond symbols)and 40 mM CaCl₂ (square symbols) current data at two voltage protocols(panels A and C, stepping from zero mV; and B and D, stepping from −120mV) for Solas (panels A and B) and RNS-60 (panels C and D). The resultsindicate that both Solas and RNS-60 solutions activated acalcium-induced non-linear whole cell conductance. The effect wasgreater with RNS-60 (indicating a dosage responsiveness), and withRNS-60 was only increased at higher calcium concentrations. Moreover,The non-linear calcium dependent conductance at higher calciumconcentration was also increased by the voltage protocol.

The data of this second set of experiments further indicates an effectof RNS-60 saline and Solas saline for whole cell conductance dataobtained in Calu-3 cells. The data indicate that 15-min incubation witheither saline produces a distinct effect on the whole cell conductance,which is most evident with RNS-60, and when external calcium isincreased, and further indicates that the RNS-60 saline increases acalcium-dependent non-linear component of the whole cell conductance.

The accumulated evidence suggests activation by Revalesio saline of ionchannels, which make different contributions to the basal cellconductance.

Taken together with Applicants' other data (e.g., the data of Applicantsother working Examples) particular aspects of the present inventionprovide compositions and methods for modulating intracellular signaltransduction, including modulation of at least one of membranestructure, membrane potential or membrane conductivity, membraneproteins or receptors, ion channels, lipid components, or intracellularcomponents with are exchangeable by the cell (e.g., signaling pathways,such as calcium dependant cellular signaling systems, comprising use ofthe inventive electrokinetically generated solutions to impartelectrochemical and/or conformational changes in membranous structures(e.g., membrane and/or membrane proteins, receptors or other membranecomponents) including but not limited to GPCRs and/or g-proteins.According to additional aspects, these effects modulate gene expression,and may persist, dependant, for example, on the half lives of theindividual messaging components, etc.

Example 25 Atomic Force Microscopy (AFM) Measurements of the InventiveElectrokinetic Fluid (RNS-60) Indicated the Presence and/or Formation ofHydrophobic Surface Nanobubbles that were Substantially Smaller thatThose Present in Control ‘Pressure Pot’ (PNS-60) Fluid

Overview. Applicants used Atomic Force Microscopy (AFM) measurements tocharacterize hydrophobic nanobubbles in the inventive electrokineticfluid (RNS-60).

Materials and Methods:

AFM studies. AFM studies were preformed at an art-recognized NanotechUser Facility (NTUF). For AFM studies, a very small and sensitive needleis dipped into a droplet of water placed onto a hydrophobic surface. Theneedle then scans over the water/surface interface at rates such as 1mm² in ˜15 minutes. The needle records any imperfections in the surfacegeometry, and is sensitive enough to record the presence of smallbubbles.

The Silicon substrate upon which the water droplets were placed wasprepared using Trichloro(1H,1H,2H,2H-perfluorooctyl)silane), and theresulting hydrophobic surface causes water to bead up with contactangles of approximately 95 degrees. This coating is used in many AFMstudies, in part, because it is particularly durable.

Solution Preparation. Two test solutions were studied: RNS-60 andPNS-60. RNS-60 is an inventive electrokinetic fluid comprising 60 ppmoxygen, whereas PNS-60 is a non-electrokinetic control fluid comprising60 ppm oxygen prepared by conventional exposure to a pressurized oxygenhead (i.e., pressure pot oxygenated fluid). Each test solution wasinitially buffered by addition of a small amount of neutral phosphatebuffer (pH 7) solution, and approximately 60-70 uL of each buffered testsolution (approximately 22° C.) was placed onto a previously preparedsilica plate.

Results:

Under AFM, the RNS-60 droplet displayed a distribution of about 20hydrophobid nanobubbles in a 1 mm² area, having dimensions of 20 nm wideand 1.5 nm tall or smaller (FIG. 121A). By contrast, under AFM, thePNS-60 droplet displayed approx 5 hydrophobic nanobubbles in a 1 mm²area, having dimensions of 60 nm wide and 5 nm tall (FIG. 121B). ThePNS-60 droplet, therefore, had much fewer and much larger hydrophobicnanobubbles compared to the RNS60 droplet.

According to particular aspects, therefore, there is a substantialdifference in the size and distribution of hydrophobic surfacenanobubbles between the RNS-60 and PNS-60 test solutions, where thenanobubbles are either initially present in, and/or formed within thetest fluids during AFM measurement.

As discussed elsewhere herein, according to particular aspects of thepresent invention, the inventive electrokinetically altered fluidscomprise an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter of less than about 100 nanometers and stably configured in theionic aqueous fluid in an amount sufficient to provide, upon contact ofa living cell by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity.

Applicants point out, however, that the hydrophobic bubbles (forming ona hydrophobic surface), such as those observed in AFM experiments arelikely fundamentally different from inventive biologically-activecharge-stabilized nanostructure disclosed herein. According toparticular aspects therefore, while the AFM experiments in this workingExample support, based on the size and distribution hydrophobic bubbleformation, that the inventive electrokinetic fluids (e.g., RNS-60) arefundamentally distinct from non-electrokinetic control fluids, thehydrophobic bubbles are likely distinct from and/or derived from theinventive charge-stablilized oxygen-containing nanostructures describedin detail elsewhere herein. In any event, relative to the inventiveelectrokinetic fluids, control pressure pot oxygenated fluids do notcomprise charge-stabilized oxygen-containing nanostructures capable ofmodulation of at least one of cellular membrane potential and cellularmembrane conductivity.

1. A electrokinetically-altered fluid composition, comprising anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized gas-containing nanostructuressubstantially having an average diameter of less than about 100nanometers and stably configured in the ionic aqueous fluid in an amountsufficient to provide, upon contact of a living cell by the fluid,modulation of at least one of cellular membrane potential and cellularmembrane conductivity.
 2. The electrokinetically-altered fluidcomposition of claim 1, wherein charge-stabilized oxygen-containingnanostructures are the major charge-stabilized gas-containingnanostructure species in the fluid.
 3. The electrokinetically-alteredfluid composition of claim 1, wherein the charge-stabilizedgas-containing nanostructures comprise charge-stabilizednitrogen-containing nanostructures
 4. The electrokinetically-alteredfluid composition of claim 2, wherein the percentage of dissolved oxygenmolecules present in the fluid as the charge-stabilizedoxygen-containing nanostructures is a percentage selected from the groupconsisting of greater than: 0.01%, 0.1%, 1%, 5%; 10%; 15%; 20%; 25%;30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; and95%.
 5. The electrokinetically-altered fluid composition of claim 1,wherein the total dissolved gas is substantially present in thecharge-stabilized gas-containing nanostructures.
 6. Theelectrokinetically-altered fluid composition of claim 1, wherein thecharge-stabilized gas-containing nanostructures substantially have anaverage diameter of less than a size selected from the group consistingof: 90 nm; 80 nm; 70 nm; 60 nm; 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; andless than 5 nm.
 7. The electrokinetically-altered fluid composition ofclaim 1, wherein the ionic aqueous solution comprises a saline solution.8. The electrokinetically-altered fluid composition of claim 1, whereinthe fluid is super-gasified.
 9. The electrokinetically-altered fluidcomposition of claim 1, wherein the charge-stabilized gas-containingnanostructures comprise at least one positively-charged ion selectedfrom the group consisting of alkali metal based salts including Li+,Na+, K+, Rb+, and Cs+, alkaline earth based salts including Mg++ andCa++, and transition metal-based positive ions including Cr, Fe, Co, Ni,Cu, and Zn, in each case along with any suitable counterioniccomponents.
 10. The electrokinetically-altered fluid composition ofclaim 1, wherein the fluid comprises at least one of a form of solvatedelectrons, and an electrokinetically modified or charged oxygen species.11. The electrokinetically-altered fluid composition of claim 10,wherein the form of solvated electrons or electrokinetically modified orcharged oxygen species are present in an amount of at least 0.01 ppm, atleast 0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, atleast 5 ppm, at least 7 ppm, at least 10 ppm, at least 15 ppm, or atleast 20 ppm.
 12. The electrokinetically-altered fluid composition ofclaim 10, wherein the electrokinetically-altered fluid comprises a formof solvated electrons stabilized, at least in part, by molecular oxygen.13. The electrokinetically-altered fluid composition of claim 1, whereinthe ability to modulation of at least one of cellular membrane potentialand cellular membrane conductivity persists for at least two, at leastthree, at least four, at least five, at least 6, at least 12 months, orlonger periods, in a closed gas-tight container.
 14. Theelectrokinetically-altered fluid composition of claim 1, whereinalteration of the electrokinetically altered aqueous fluid comprisesexposure of the fluid to hydrodynamically-induced, localizedelectrokinetic effects.
 15. The electrokinetically-altered fluidcomposition of claim 14, wherein, exposure to the localizedelectrokinetic effects comprises exposure to at least one of voltagepulses and current pulses.
 16. The electrokinetically-altered fluidcomposition of claim 14, wherein the exposure of the fluid tohydrodynamically-induced, localized electrokinetic effects, comprisesexposure of the fluid to electrokinetic effect-inducing structuralfeatures of a device used to generate the fluid.
 17. Theelectrokinetically-altered fluid composition of claim 1, whereinmodulation of at least one of cellular membrane potential and cellularmembrane conductivity comprises altering of a conformation, ligandbinding activity, or a catalytic activity of a membrane associatedprotein.
 18. The electrokinetically-altered fluid composition of claim17, wherein the membrane associated protein comprises at least oneselected from the group consisting of receptors, transmembranereceptors, ion channel proteins, intracellular attachment proteins,cellular adhesion proteins, integrins, etc.
 19. Theelectrokinetically-altered fluid composition of claim 18, wherein thetransmembrane receptor comprises a G-Protein Coupled Receptor (GPCR).20. The electrokinetically-altered fluid composition of claim 19,wherein the G-Protein Coupled Receptor (GPCR) interacts with a G proteina subunit.
 21. The electrokinetically-altered fluid composition of claim20, wherein the G protein α subunit comprises at least one selected fromthe group consisting of Gα_(s), Gα_(i), Gα_(q), and Gα₁₂.
 22. The melectrokinetically-altered fluid composition of claim 21, wherein the atleast one G protein α subunit is Gα_(q).
 23. Theelectrokinetically-altered fluid composition of claim 20, whereinmodulation of at least one of cellular membrane potential and cellularmembrane conductivity, comprises modulating whole-cell conductance. 24.The electrokinetically-altered fluid composition of claim 23, whereinmodulating whole-cell conductance, comprises modulating at least one ofa linear or non-linear voltage-dependent contribution of the whole-cellconductance.
 25. The electrokinetically-altered fluid composition ofclaim 1, wherein modulation of at least one of cellular membranepotential and cellular membrane conductivity comprises modulation of acalcium dependant cellular messaging pathway or system.
 26. Theelectrokinetically-altered fluid composition of claim 1, whereinmodulation of at least one of cellular membrane potential and cellularmembrane conductivity comprises modulation of phospholipase C activity.27. The electrokinetically-altered fluid composition of claim 1, whereinmodulation of at least one of cellular membrane potential and cellularmembrane conductivity comprises modulation of adenylate cyclase (AC)activity.
 28. The electrokinetically-altered fluid composition of claim1, wherein the electrokinetically-altered aqueous fluid comprisesdissolved gas in an amount of at least 8 ppm, at least 15, ppm, at least25 ppm, at least 30 ppm, at least 40 ppm, at least 50 ppm, or at least60 ppm oxygen at atmospheric pressure.
 29. Theelectrokinetically-altered fluid composition of claim 28, wherein thegas in the charge-stabilized nanostructures of the fluid or solution ispresent in an amount of at least 25 ppm.
 30. A method of producing anelectrokinetically-altered aqueous fluid or solution, comprising:providing a flow of a fluid material between two spaced surfaces inrelative motion and defining a mixing volume therebetween, wherein thedwell time of a single pass of the flowing fluid material within andthrough the mixing volume is greater than 0.06 seconds or greater than0.1 seconds; and introducing gas into the flowing fluid material withinthe mixing volume under conditions suitable to dissolve at least 20 ppm,at least 25 ppm, at least 30, at least 40, at least 50, or at least 60ppm gas into the material, and electrokinetically alter the fluid orsolution, wherein an electrokinetically altered aqueous fluid comprisingan ionic aqueous solution of charge-stabilized gas-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide, upon contact of a living cell by thefluid, modulation of at least one of cellular membrane potential andcellular membrane conductivity is provided.
 31. The method of claim 30,wherein the gas is infused into the material in less than 100milliseconds, less than 200 milliseconds, less than 300 milliseconds, orless than 400 milliseconds.
 32. An electrokinetically altered gasifiedaqueous fluid or solution made according to any one of claims 30 and 31.33. A method of producing an electrokinetically altered aqueous fluid orsolution, comprising: providing a flow of a fluid material between twospaced surfaces defining a mixing volume therebetween; and introducinggas into the flowing material within the mixing volume under conditionssuitable to infuse at least 20 ppm, at least 25 ppm, at least 30, atleast 40, at least 50, or at least 60 ppm gas into the material in lessthan 100 milliseconds, less than 200 milliseconds, less than 300milliseconds, or less than 400 milliseconds, to electrokinetically alterthe fluid or solution, wherein an electrokinetically altered aqueousfluid comprising an ionic aqueous solution of charge-stabilizedgas-containing nanostructures substantially having an average diameterof less than about 100 nanometers and stably configured in the ionicaqueous fluid in an amount sufficient to provide, upon contact of aliving cell by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity is provided. 34.The method of claim 33, wherein the dwell time of the flowing materialwithin the mixing volume is greater than 0.06 seconds or greater than0.1 seconds.
 35. The method of any one of claims 30 through 34, whereinthe ratio of surface area to the volume is at least 12, at least 20, atleast 30, at least 40, or at least
 50. 36. An electrokinetically alteredgasified aqueous fluid or solution made according to any one of claims33 and
 34. 37. A method of producing an electrokinetically-alteredaqueous fluid or solution, comprising use of a mixing device forcreating an output mixture by mixing a first material and a secondmaterial, the device comprising: a first chamber configured to receivethe first material from a source of the first material; a stator; arotor having an axis of rotation, the rotor being disposed inside thestator and configured to rotate about the axis of rotation therein, atleast one of the rotor and stator having a plurality of through-holes; amixing chamber defined between the rotor and the stator, the mixingchamber being in fluid communication with the first chamber andconfigured to receive the first material therefrom, and the secondmaterial being provided to the mixing chamber via the plurality ofthrough-holes formed in the one of the rotor and stator; a secondchamber in fluid communication with the mixing chamber and configured toreceive the output material therefrom; and a first internal pump housedinside the first chamber, the first internal pump being configured topump the first material from the first chamber into the mixing chamber,to electrokinetically alter the fluid or solution, wherein anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized gas-containing nanostructuressubstantially having an average diameter of less than about 100nanometers and stably configured in the ionic aqueous fluid in an amountsufficient to provide, upon contact of a living cell by the fluid,modulation of at least one of cellular membrane potential and cellularmembrane conductivity is provided.
 38. A method of producing anelectrokinetically altered gasified aqueous fluid or solution,comprising use of a mixing device for creating an output mixture bymixing a first material and a second material, the device comprising: astator; a rotor having an axis of rotation, the rotor being disposedinside the stator and configured to rotate about the axis of rotationtherein; a mixing chamber defined between the rotor and the stator, themixing chamber having an open first end through which the first materialenters the mixing chamber and an open second end through which theoutput material exits the mixing chamber, the second material enteringthe mixing chamber through at least one of the rotor and the stator; afirst chamber in communication with at least a majority portion of theopen first end of the mixing chamber; and a second chamber incommunication with the open second end of the mixing chamber, toelectrokinetically alter the fluid or solution, wherein anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized gas-containing nanostructuressubstantially having an average diameter of less than about 100nanometers and stably configured in the ionic aqueous fluid in an amountsufficient to provide, upon contact of a living cell by the fluid,modulation of at least one of cellular membrane potential and cellularmembrane conductivity is provided.
 39. The method of claim 36, 37wherein the first internal pump is configured to impart acircumferential velocity into the first material before it enters themixing chamber.
 40. The method of claim 1, wherein the charge-stabilizedoxygen-containing nanostructures of the electrokinetically-altered fluidcomprise at least one salt or ion from Tables 1 and 2 disclosed herein.